TWI284042B - A composition and method for the enhancement of the efficacy of drugs - Google Patents

Abstract

The present invention relates to enhancement of the efficacy of drugs, and more particularly, with overcoming the resistance of cells or organisms to drugs. In particular, the present invention provide a method for enhancing the effectiveness of a cytotoxic or anti-neoplastic agent, comprising the step of co-administering said agent with hyaluronan wherein co-administration with hyaluronan enhances the agents' cancer cell-killing potential.

Description

經濟部智慧財產局員工消費合作社印製 1284042 A7 ______B7__ 五、發明説明（） 1 發明領斑 本發明係有關一種藥物之有效性之增強，更特別的 是，其具有克服細胞或生物體對於藥物之抗性。 發明背景 很多化學治療劑或藥物之臨床上的有用性嚴重地受到 對該藥物之細胞抗性的出現所影響。因此，相當多的研究 已在進行，而嘗試著說明涉及抗藥性之細胞機制以及克服 該抗性的方法。至今提出許多涉及抗藥性之推定性的細胞 機制。其等包含 (i)改變藥物的代謝，其可包含降低活化作用或增加 活化作用； (ii)標的細胞或生物體對該活性化合物的不通透性； (i i i)改變一受制抑之酵素專一性； (iv) 增加標的分子生成； (v) 增高細胞毒性損害的修復；以及 (vi) 利用另一生物醫學路徑而繞過該受抑制的反應。 在藥物抗性中所涉及的細胞機制是複雜的，在發展出 克服藥物抗性問題上之一般性可應用的方法，結果必然是 顯少有進步的。另一個複雜的因素是，雖然藥物抗性是一 與幾乎所有化學治療應用上有相關的問題，但其常常是與 該等需要以許多同時使用的藥物持續且長期地治療之疾病 相關。 在癌症的治療中，例如，常可觀察到癌細胞對活性化 合物之不通透性。再者，亦常發現，對一藥物的抗性可造 -4-Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Printing 1240442 A7 ______B7__ V. Invention Description (1) Invention The present invention relates to the enhancement of the effectiveness of a drug, and more particularly to overcoming cells or organisms for drugs. Resistance. BACKGROUND OF THE INVENTION The clinical usefulness of many chemotherapeutic agents or drugs is severely affected by the appearance of cellular resistance to the drug. Therefore, considerable research has been conducted, and attempts have been made to explain the cellular mechanisms involved in drug resistance and methods for overcoming the resistance. Many presumed cellular mechanisms involving drug resistance have been proposed to date. These include (i) altering the metabolism of the drug, which may include reducing activation or increasing activation; (ii) impermeability of the subject cell or organism to the active compound; (iii) altering a constitutive enzyme specificity (iv) increase the target molecule production; (v) increase the repair of cytotoxic damage; and (vi) bypass the inhibited response using another biomedical pathway. The cellular mechanisms involved in drug resistance are complex, and in general, methods that have been developed to overcome drug resistance problems have inevitably been less advanced. Another complicating factor is that while drug resistance is a problem associated with almost all chemotherapeutic applications, it is often associated with such diseases that require long-term treatment with many concurrent drugs. In the treatment of cancer, for example, cancer cells are often observed to be impermeable to active compounds. Furthermore, it is often found that resistance to a drug can be made -4-

• In HI m !ΊΙ^-----Φ丨丨 (請先閱讀背面之注意事項再填寫本頁) 、1Τ 1284042 A7 B7 五、發明説明（） 2 成對另一生化上完全不同的藥物之抗性。此已被稱作多種 抗藥性。一般受到多重抗藥性影響的藥物包含有阿黴素 (亞德里亞黴素）、長春新驗（vincristine)、長春花驗 (vinblastine)、秋水仙素（c〇lchicine)與放線菌素 〇。 至少在某些例子中，多重抗藥性係為複雜的表型，其係 與被稱為MdrI蛋白質（即已知的P_醣蛋白）之細胞膜藥物 送出輸送子（efflux-transporter)的高位準的表現相關 連。此一膜’’泵”具有廣的專一性，且作用以自細胞去除 廣範圍之各式各樣的化學上不相關的毒素（參見 Endicott et al·，1 989) ° 近來，對一些微生物而言，一廣範圍之抗藥性相似機 制已被報告。該等結果顯示出相當廣範圍受質專一性的細 菌送出系統的存在，此是相似於於哺乳動物細胞之多重抗 藥性泵（參見Nikaido，1 993)。 该逆轉抗藥性之物質已知為抗性修飾劑（RM a s)。 在該等人類腫瘤對之具有抗性的化學治療劑具細胞毒性上 是重要的。雖然許多試劑已被定為在人工環境下rmAs， 但此等試劑的大部份具有低治療潛力或不具有治療潛力， 盍因其需要逆轉多重抗藥性的劑量下是在活體内具高毒性 的。例如，代謝毒素，例如疊氮化物，係能夠在活體外逆 轉抗藥性，但在活體内則無有用處。大部分其它高效用的 RMAs，例如，PSC83 3，顯然係作為一位蛋白質 上之藥物結合址的競爭性拮抗性。許多該等試劑亦具有毒 (請先閲讀背面之注意事項再填寫本頁) 、1Τ 經濟部智慧財產局員工消費合作社印製• In HI m !ΊΙ^-----Φ丨丨 (please read the notes on the back and fill out this page), 1Τ 1284042 A7 B7 V. Inventions () 2 Pairs of another biochemically completely different drug Resistance. This has been referred to as multiple drug resistance. Drugs generally affected by multiple drug resistance include doxorubicin (adriamycin), vincristine (vincristine), vinblastine (vinblastine), colchicine (c〇lchicine) and actinomycin. In at least some instances, multidrug resistance is a complex phenotype that is associated with a high level of cell membrane drug delivery (efflux-transporter) called MdrI protein (known as P-glycoprotein). Performance related. This membrane ''pump' has a wide range of specificities and acts to remove a wide range of chemically unrelated toxins from cells (see Endicott et al., 1 989). Recently, for some microorganisms A wide range of drug resistance-like mechanisms have been reported. These results show the existence of a fairly broad range of plasmid-specific bacterial delivery systems, which are multi-drug resistant pumps similar to mammalian cells (see Nikaido, 1 993). The substance that reverses resistance is known as a resistance modifier (RM as). The chemotherapeutic agent to which these human tumors are resistant is cytotoxic, although many reagents have been determined. For rmAs in artificial environments, but most of these agents have low therapeutic potential or no therapeutic potential, and are highly toxic in vivo due to their need to reverse multiple drug resistance. For example, metabolic toxins, For example, azide can reverse resistance in vitro, but it is not useful in vivo. Most other high-efficiency RMAs, for example, PSC83 3, are apparently a single protein. Competitive antagonist of drug binding sites. Many of these agents also have toxic (Please read the notes on the back of this page to fill out), 1Τ Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed

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經濟部智慧財產局員工消費合作社印製 性，此限制其等在活體内的有用性。結果，有—種發展出 用以逆轉多重抗藥性的另一藥理策略。 在嘗試去克服抗贅生藥劑之腫瘤攝取不佳以及降低系 統性毒性（Singh et al·，1991)，調查家已試著將抗贅 生藥劑，例如，氨甲喋呤（MTX)，鎖定於惡性組織的位 置 些研究亦已嘗試，利用將鏈結藥物鏈結至一依自身 對腫瘤的親和性而被選定之聚合物，而將化學治療試鎖定 於腫瘤（Hudecz et al·，1 993; Klein et al·，1 994; Ahma et al·，1 996)。然而，雖然這些聚合物可有助於 將活性藥物輸送至該等鎖定組織，但其並不必然地會克 服該等藥物的抗性問題。 已被建議可作為腫瘤鎖定劑的一種聚合物係為透明 夤（Η A)。Η A，亦為已知的透明質酸，係為一天然存在 之包含一線性鏈聚合物的多醣，其被發明在所有的動物領 域中皆存在有。HA係為高度水溶性，此使其成為一供生 物系統用之理想藥物傳送載劑。 申請人很吃驚地發現，HA顯示出獨特的結構性以及 生理化學性特性，該等特性不僅可促住其作為藥物載劑的 使用性，同時亦有助於克服抗藥性。在高濃度的〜 lg/L，Η A採用一硬化的隨機捲繞構型，該構型相對於分 子質量佔據一另外的體積（Laurent，1 970)，且在此位準 或低於該位準，其對巨分子網絡形成一疏鬆鏈結（第工 圖）。雖然不希望為任何理論所限制，但我們考量，美於 -6- 本紙張尺度適用中關家標準（CNS ) A4規格（21GX297公董〉 ------- 丨 n K---Φ------------0—II, (請先閲讀背面之注意事項再填寫本頁) 0 —^^1 —^ϋ n ϋ-i 1284042 經濟部智慧財產局員工消費合作社印製 A7 五、發明説明（ 该Η A該物理特性，在多醣與試劑之間的相互作用機構， 諸如’氨甲喋呤，可能是採取一種或二種形式： (i)化學相互作用（第2A圖）。 離子鍵結可能發生於MTX胺基與HA的羧基，但如此 相互作用作造成沈澱。另外可能的相互作用是經由在藥物 上可使用的胺基與在Η A之羥基二者間的氫鍵結作用，但 此不太可能是因為氨甲喋吟在水中是相當不溶的，因此， 若是有此現象發生的話，其應該是一十分弱的相互作用。 在MTX與HA二者間最可能的鍵結是經由MTX，S多數個 疏水性基團與HA之二級結構中的疏水性區點（patch)二 者間的疏水性相互作用（S c 〇 11 e t a 1.，1 9 8 9 )。 (Π)分子結合 當Μ Τ X在Η Α凝膠中僅係為混合型（第2 Β圖）而無特 定化學鍵結形成，MTX可夾帶於一由高濃度HA所形成的 三維篩網内（Mikelsaar與Scott，1 994)，而使得該藥物 在投藥後僅由該ΗA中擴散出來。若HA係被快速地攝取 且為特定細胞受體所結合，則該藥物將會在該等點，例 如，淋巴節、肝、骨髓、具有HA受體之腫瘤細胞，以高 度濃釋放出來。 雖然’再次希望不要被任何特定的理論所限制，但 Η A可以幫助鎖定活性劑的機制可能是經由在一些腫瘤細 胞上HA受體之過度表現（Stamenkovic et al.，1991. \^1^6七&1.，1998)。該11八受體€044，透明質調節 移動性受體（RHAMM)與ICAM-1，已被連結至腫瘤的The Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative prints, which limits its usefulness in vivo. As a result, another pharmacological strategy has been developed to reverse multiple drug resistance. In an attempt to overcome the poor tumor uptake of anti-neoplastic agents and to reduce systemic toxicity (Singh et al., 1991), investigators have tried to lock anti-neoplastic agents, such as methotrexate (MTX), into malignant tissue. Location studies have also attempted to target chemotherapeutic trials to tumors by linking the linked drug to a polymer selected for its own affinity for the tumor (Hudecz et al., 993; Klein et al) ·, 1 994; Ahma et al., 1 996). However, while these polymers may aid in the delivery of the active drug to the locked tissues, they do not necessarily overcome the resistance problems of such drugs. A polymer that has been suggested as a tumor locking agent is transparent Η (Η A). Η A, also known as hyaluronic acid, is a naturally occurring polysaccharide comprising a linear chain polymer which was invented in all animal domains. HA is highly water soluble, making it an ideal drug delivery vehicle for use in biosystems. Applicants have surprisingly found that HA exhibits unique structural and physiochemical properties that not only promote its use as a drug carrier, but also help overcome drug resistance. At high concentrations of ~ lg/L, Η A uses a hardened random-wound configuration that occupies an additional volume relative to the molecular mass (Laurent, 1 970), and at or below this level Quasi, it forms a loose chain (the map) of the giant molecular network. Although we do not wish to be bound by any theory, we consider that the beauty of the paper scale is applicable to the Chinese National Standard (CNS) A4 specification (21GX297 director) ------- 丨n K---Φ ------------0-II, (Please read the notes on the back and fill out this page) 0 —^^1 —^ϋ n ϋ-i 1284042 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumption Cooperative Printed A7 V. Description of the invention (The physical properties of the ΗA, the interaction mechanism between the polysaccharide and the reagent, such as 'metamidine, may take one or two forms: (i) chemical interaction (Fig. 2A) Ionic bonding may occur at the MTX amine group and the carboxyl group of HA, but interacts to cause precipitation. Another possible interaction is via a chemically usable amine group and a hydrogen bond between the hydroxyl groups of Η A. Junction, but this is unlikely because methotrexate is quite insoluble in water, so if this happens, it should be a very weak interaction. Most likely between MTX and HA The bond is the hydrophobicity of the secondary structure of most of the hydrophobic groups via MTX, S Patching the hydrophobic interaction between the two (S c 〇11 eta 1., 1 9 8 9 ). (Π) Molecular binding when Μ Τ X is only mixed in the Η Α gel (2nd) Without specific chemical bonding, MTX can be entrained in a three-dimensional screen formed by high concentrations of HA (Mikelsaar and Scott, 1994), allowing the drug to diffuse only from the sputum A after administration. If the HA line is rapidly taken up and bound by a specific cellular receptor, the drug will be released at a high concentration at these points, for example, lymph nodes, liver, bone marrow, and tumor cells with HA receptors. 'I hope again not to be bound by any particular theory, but the mechanism by which A can help lock in the active agent may be through excessive expression of HA receptors on some tumor cells (Stamenkovic et al., 1991. \^1^6 7 &1., 1998). The 11-receptor €044, the hyaluronan-regulated mobile receptor (RHAMM) and ICAM-1, have been linked to tumors.

—訂 (請先閱讀背面之注意事項再填寫本頁) _·· 1284042 A7 --- —_____B7 五、發明説明（） 5 起因（Bartolazzi et al·，1 994)與發展（GUnthert 1 993; Arch et al·，1 992)。RHAMM在調節腫瘤細胞 移動性與侵入性上是主要的因子（Hardwick et al·， 1 992)。已顯示出RHAMM對纖維組織母細胞之H-ras 轉形作用是需要的（Hall et al·，1 995)，而此會使得該 觉體為腫瘤形成與生長的一可能的參與者。I cam-i， 一暫時結合至Η A代謝上的受體（μ c C ourt e t al ·， 1 9 9 4) ’疋在經轉形的組織中’例如，小鼠肥胖細胞瘤 中，以及（Gustafson et al·，1 995a)，且在人類乳癌 腫瘤之腫瘤細胞基質（strolna )與集群（〇gawa et al*，35 1998)，是被高度地表現出的。 腫瘤細胞之Η A受體的增加表現，提供一種供試著將 Η A併入化學治療之療程（r ^ g丨m e n s)的基本理由。但是， 至今所獲得之有限資料教示方向是與現今所請求方法相背 離。例如，利用將ΗA化學複合至絲裂黴素C與艾普比新 (epirubicin )已可獲得有限的成功；調查家係能抑制直 腸癌之生長達 0.8-25% (Akima et α/·，1996)。—Set (please read the notes on the back and fill out this page) _·· 1284042 A7 --- —_____B7 V. Description of invention () 5 Causes (Bartolazzi et al·, 994) and development (GUnthert 1 993; Arch Et al., 1 992). RHAMM is a major factor in regulating tumor cell mobility and invasiveness (Hardwick et al., 1992). It has been shown that RHAMM is required for H-ras transduction of fibroblasts (Hall et al., 995), which would make the subject a potential participant in tumor formation and growth. I cam-i, a receptor that transiently binds to ΗA metabolism (μ c C ourt et al ·, 1 9 9 4) '疋 in transformed tissue', eg, in mouse obese cell tumors, and (Gustafson et al., 995a), and is highly expressed in the tumor cell stroma and cluster of human breast cancer tumors (〇gawa et al*, 35 1998). The increased performance of A receptors in tumor cells provides a rationale for trying to incorporate Η A into the course of chemotherapy (r ^ g丨m e n s). However, the limited information teaching directions obtained so far are contrary to the methods currently requested. For example, the limited success of chemically complexing ΗA to mitomycin C and epirubicin; investigating families can inhibit the growth of rectal cancer by 0.8-25% (Akima et α/·, 1996) ).

Klein與其同僚（1994)報告出經移植老鼠乳腺與費雪膀 光癌之藥物的提高攝取，可僅利用將Η Α與5 - F U混合來 達成。小鼠肥胖細胞瘤已顯示出對靜脈注射的Η A具有一 親和性（Gustafson et α/·，1995b)。 然而，雖有些HA的研究已顯示，HA可作為藥物的載 劑，但該等研究無有一者顯示，ΗA係能夠克服對藥物之 細胞抗性。 -8 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公羡) " ' 丨tLl·-——4! (請先閱讀背面之注意事項再填寫本頁} 訂 經濟部智慧財產局員工消費合作社印製 1284042 A7Klein and his colleagues (1994) reported increased intake of transplanted mouse mammary gland and Fisher's light cancer drugs, which can be achieved by simply mixing Η and 5 - F U. Mouse obese cell tumors have been shown to have an affinity for intravenously injected Η A (Gustafson et α/, 1995b). However, although some HA studies have shown that HA can be used as a drug carrier, none of these studies have shown that ΗA line can overcome cell resistance to drugs. -8 - This paper scale applies to China National Standard (CNS) A4 specification (210X297 public) " ' 丨tLl·-——4! (Please read the notes on the back and fill out this page) Employee consumption cooperative printed 1240442 A7

畳Jlfe要— 本發明係提供一種增進細胞毒性劑或抗贅生劑的有效 、、法 /、包3下列步驟··將該藥物與透明質共投藥給 該試劑’其中該透明質的共投藥可促進該試劑之殺滅癌細 胞的效用。 般而5 ’該細胞毒性劑或抗贅生劑與透明質的共投 某可促進該忒劑之殺癌細胞的效用。亦即，一般抗藥性的 癌細胞會變得對其敏感。 較佳的是，該藥物係為氨甲喋呤、佩里塔克爾 (Paclitaxel)(紅豆杉醇）或5氟尿哺咬（5_pu)。 依據本發明的第二方面，其係提供一種細胞毒性或抗 头生|±藥學組成物’其包含有透明質與_細胞毒性劑或抗 %生劑。可擇任地是，傳統的藥學佐劑亦可被包含在該藥 學組成物中。 一般而言’該藥物係被夾帶在透明質内或被結合至該 透明質上。 經濟部智慧財產局員工消費合作社印製 依據本本發明的第三方面，其係提供一種增強細胞毒 f生州或抗夤生劑之效力的方法，其包含投藥以一包含有該 透明質與該藥劑的藥學組成物。 雖然不想要被該理論所束縛，但一般相信，用來克服 抗藥性的可能機制係為： 1*透明質結合至位在抗藥性細胞受體上或經由大量的細 胞内呑作用（endocytosis)而進入細胞，因而造成被夾帶畳Jlfe-- The present invention provides an effective method for improving cytotoxic or anti-neoplastic agents, and/or the following steps: · co-administering the drug with hyaluronic acid to the agent, wherein the hyaluronan is co-administered It can promote the effect of the agent to kill cancer cells. In general, a co-injection of the cytotoxic or anti-neoplastic agent with hyaluronic acid promotes the efficacy of the elixir to kill cancer cells. That is, generally resistant cancer cells become sensitive to them. Preferably, the drug is methotrexate, Paclitaxel (taxol) or 5-fluorourine (5_pu). According to a second aspect of the present invention, there is provided a cytotoxic or anti-head-to-medicine composition comprising a hyaluronic acid-to-cytotoxic agent or an anti-reagent. Alternatively, a conventional pharmaceutical adjuvant may also be included in the pharmaceutical composition. Generally, the drug is entrained within or incorporated into the hyaluronic substance. According to a third aspect of the present invention, there is provided a method for enhancing the efficacy of a cytotoxic or anti-neoplastic agent, comprising administering a hyaluronic acid comprising the hyaluronic acid a pharmaceutical composition of a medicament. Although not wishing to be bound by this theory, it is generally believed that the possible mechanisms for overcoming drug resistance are: 1* hyalin binding in place on drug-resistant cellular receptors or via large amounts of intracellular endocytosis Entering the cell, thus causing entrainment

1284042 A7 ------ -B7 __五、發明説明（） 7 或結合的藥劑被輸送入細胞内，而容許其變得具有治療活 性。 2 ·透明質結合抗藥性細胞之表面，其中該被夾帶或結合 的藥劑由篩網擴散到細胞，因而造成藥物被輸送至抗藥性 細胞。 3·透明質與其它黏多醣採用一種可夾帶該藥劑之盤繞的 構型’且亦可維合各種藥劑。 因此，在第四方面，本發明係提供一種降低或克服抗 藥性的方法，其包含共投藥細胞毒性劑或抗贅生劑與一黏 多醣一起的步驟，該黏多醣係能夠夾帶以及/或結合該藥 劑，且能夠結合至位在該抗藥性留有上的受體或經由大 的細胞内吞作用而進入細胞，其中該藥劑係被輸送至該 胞内，藉此使得該藥劑變得具有治療活性。 雖然並不想被理論所束縛，但亦可能的是，該透明 與一藥劑的組合會造成該藥物係被留滯在該留有中達一 較長的時間，而容許-延長的釋放作用與較多時間供該 物發揮其藥理效用。 因此，本發明的第五方面，係為 宁馬徒供一種增強一藥物 之效力的方法，其包含將該藥劑與_ 黏多醣一起投藥的步 驟，該黏多聽係與該藥物以一方式相結合，而使得 係被留滯在該細胞中達一段比單獨投予該藥物更長的期 間。例如，當該藥物係為細胞毒性 ,好〜“、去 性樂劑時’該藥物係被留 滞在該細胞中達一段較長的時間，& „ 知 各昨一延長的釋放作用 與較多時間供該藥物發揮其藥理效用。 评從作用 -10 量 質 段藥 物 留 ---讀丨丨 (請先閱讀背面之注意事項再填寫本頁) l· 訂 1·- ¥紙張尺度適用中國國家摞準（CNS ) A4規格（210X2^5 -___ f : ----------/ 1284042 A7 _ ______Β7 五、發明説^^) ~" ----— 8 依據本發明的第六方面，則是提供—種治療抗藥性疾 病的方法，其包含將透明質與-藥物共投藥至需要該治療 之病人的步驟。 在-實施例中，本發明的此方面係提供一種治療抗藥 性疾病的方法，其包含有-將包含有透明質與一藥物的組 成物投藥給需要該治療的病人之步驟。 更特別的是，用以治療抗藥性疾病昀方法是可應用至 具有抗藥性癌症的病人。較佳的是該癌病係對氨甲喋呤或 5 - F U具有抗藥性。 更佳的是，該抗藥性是多重抗藥性。 依據本發明的第七方面，其係提供一種治療癌症的方 法，其包含將一藥物與黏多醣共投藥之步驟，該黏多醣係 月b夠夾帶或結合該藥物以及/或與該藥物以一方式相結合 而使得該藥物被留滯在癌細胞内達一段比該藥物單獨投藥 更長的期間。 依據本發明的第八方面，其係提供一種降低一藥物的 月腸道毒性的方法，其包含將一藥物與黏多醣共投藥之步 驟’該黏多醣係能夠夾帶或結合該藥物以及/或與該藥物 以一方式相結合而使得該藥物可降低該胃腸道毒性。 本說明書所有說明與申請專利範圍，該詞“包含，，以 及該詞的變化，例如，“包含有，，意指“含有但不限 於’且並不想要來排除其它添加劑、成份、完整事物或 步驟。 flJUSL要說明 ___— -11- 本^(CNS) A4 規格 -—- (請先閲讀背面之注意事項再填寫本頁) 、?τ 經濟部智慧財產局員工消費合作社印製 12840421284042 A7 ------ -B7 __ V. INSTRUCTIONS (7) or a combined agent is delivered into a cell to allow it to become therapeutically active. 2. The hyaluronic acid binds to the surface of the drug-resistant cell, wherein the entrained or bound agent diffuses from the mesh to the cell, thereby causing the drug to be delivered to the drug-resistant cell. 3. Hyalin and other mucopolysaccharides use a coiled configuration that entrains the agent' and can also blend various agents. Accordingly, in a fourth aspect, the present invention provides a method of reducing or overcoming drug resistance comprising the step of co-administering a cytotoxic or anti-neoplastic agent with a mucopolysaccharide capable of entrainment and/or binding The agent is capable of binding to a receptor remaining on the drug resistance or entering the cell via a large endocytosis, wherein the agent is delivered to the cell, thereby making the agent therapeutic active. Although it is not intended to be bound by theory, it is also possible that the combination of transparency and a drug will cause the drug to remain in the retention for a longer period of time, while allowing-prolonged release and comparison More time for the substance to exert its pharmacological effects. Accordingly, a fifth aspect of the present invention provides a method for enhancing the efficacy of a drug comprising the step of administering the agent together with the viscous polysaccharide, the visceral system and the drug being in a manner Binding allows the line to remain in the cell for a longer period of time than when the drug is administered alone. For example, when the drug is cytotoxic, it is good ~ ", when the sex agent is taken" the drug is left in the cell for a long period of time, & „ know each of the extended release effect and more Time for the drug to exert its pharmacological effects. Comment on the role - 10 mass segment drug retention --- read 丨丨 (please read the back of the note and then fill out this page) l· Order 1·- ¥ paper scale for China National Standard (CNS) A4 specifications (210X2 ^5 -___ f : ----------/ 1284042 A7 _ ______Β7 V. Inventions ^^) ~" -----8 According to the sixth aspect of the present invention, A method of treating a drug-resistant disease comprising the step of co-administering hyaluronan with a drug to a patient in need of such treatment. In an embodiment, this aspect of the invention provides a method of treating a drug-resistant disease comprising the step of administering a composition comprising hyaluronic acid and a drug to a patient in need of such treatment. More specifically, the method for treating drug-resistant diseases is applicable to patients with drug-resistant cancer. Preferably, the cancerous disease is resistant to methotrexate or 5-FU. More preferably, the drug resistance is multidrug resistance. According to a seventh aspect of the present invention, there is provided a method for treating cancer comprising the step of co-administering a drug with a mucopolysaccharide which is entrained or bound to the drug and/or with the drug The combination of the modes allows the drug to remain in the cancer cells for a longer period of time than when the drug is administered alone. According to an eighth aspect of the present invention, there is provided a method for reducing the intestinal toxicity of a drug comprising the step of co-administering a drug with a mucopolysaccharide capable of entraining or binding the drug and/or The drug is combined in a manner such that the drug reduces the gastrointestinal toxicity. The description of the specification and the scope of the patent application, the word "comprising," and variations of the word, for example, "includes, means "including but not limited to" and does not intend to exclude other additives, ingredients, whole things or Step: flJUSL to explain ___- -11- this ^ (CNS) A4 specifications - -- (please read the back of the note and then fill out this page), ? τ Ministry of Economic Affairs Intellectual Property Bureau employees consumer cooperatives printed 1284042

第1圖顯示，在較高濃度下，HA形成一種三維篩 網’其能夠夾帶諸如氨甲嗓呤之類的小份子。該鎖定病理 位置之HA/藥物係經由該HA快速地結合至特殊的留有受 體，而後將該藥物自該HA擴散以及/或經由該HA與/或藥 物受體之ΗA與藥物二者的共内部化而達成的。 第2A圖顯示在氨甲喋呤與ha之間可能分子相互反 應。此包含有（i)離子鍵結，（ii)氫鍵，或（iH)疏水性 鍵結。 第2B圖係為一氨甲喋呤在HA中纏結的示意圖。在較 高的濃度下，HA形成一種以大型盤繞分子為代表之三維 篩網。（*)代表氨甲喋呤，其具有僅僅454D，且十分容 易被夾帶於400-900 kD HA分子内。 第3圖顯示在裸鼠中人類乳癌腫瘤的一般病理.。第A 組顯示第II_III級人類腫瘤的形態。第B組則顯示出顯示 於第3 A圖之腫瘤的另一個切面的顯微圖。該切面顯示， 該環繞的小鼠肌肉（Μ)、腫瘤囊（C )、腫瘤壞死區域 (Ν)、浸潤腫瘤（Τ)以及（―）顯示出一已知稱為“印地 安排列（Indian files)”現象，其中該癌細胞排列成 列，此一般係與浸潤癌腫瘤相關（Carter，1 990)。 第4圖顯示乳部惡性腫瘤的傳統病理特徵。第A組顯 示一具有大區域壌死（N )之浸潤道腫瘤之特徵的切面。， 此顯示該腫瘤之較攻擊期間。第B組顯示血液細胞（_ + )的 存在，此顯示該腫瘤係接有血管且因而可存活。在相當接 近血管處，有一些可被觀察到的管道（D)。第c組顯示， -12- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） J-I：-----_衣|丨 (請先閲讀背面之注意事項再填寫本頁) 訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（） 10 被標記為（A)的細胞，如核分成片段所顯示者係，遭受到 程式化死亡（apoptosis)。 第5圖顯示於裸鼠中生長的人類乳癌腫瘤細上之Ha 受體的免疫組織化學鑑定。第A組顯示，在腫瘤中癌胚 (carcinoembryonic)抗體（CF_A)之局部化。第B組顯 示在人類乳癌瘤上的RHAMM表現。第C組顯示在人類乳 癌瘤上的IC A Μ -1的表現。該Η A受體的局部化作用， ICAM-1主要是環繞著内皮組織（_>}。第D組顯示在人類 乳癌瘤上的€〇44的表現。0〇4411係在約9 5%的腫瘤細 胞上皆可檢測得。Η :人類來源的細胞；]VI:鼠類來源的 細胞。 第6圖顯示使用作為供鎖定腫瘤之氨甲喋呤的載劑之 透明質的分子量分析結果。 第7顯示鎖定人類乳癌腫瘤之氨甲喋呤，其使用Η A 作為載劑。 第8圖顯示在HA存在下氨甲喋呤之增強攝取。 第9圖顯示當藥物與HA—起投藥時，在胃腸道中氨 甲喋呤的降低攝取。 第1 〇圖顯示在MTX/HA之間可能的物理相互作用與 其後藥物/Η A組合治療之腫瘤鎖定能力的示意表示。 第11圖顯示結合HA與5FU在活體外效用上之細胞毒 性加成性。 第12圖顯示使用HA作為載劑，5-FU鎖定人類乳癌 腫瘤。 -13- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） J-I:-----鑛II (請先閲讀背面之注意事項再填寫本頁) 訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（） 11 第13圖顯示在人類中HA的消除路徑。 第1 4圖顯示在胃、腦及肺中5 _ FU的增加攝取。 第15圖顯示在血漿5-FU藥學動力學消除上ha的效 用。 第1 6圖顯示用於定義實驗終點的標準。其係顯示標 準2 (第A組）與標準3 (第b組）。 第17圖顯示5-FU/HA佐劑治療的效用（6週的療 程）：在主要腫瘤體積上的效用。 第1 8圖顯示5-FU/HA佐劑治療的效用（6週的療 程）·在主要體重量上的效用。 第19圖顯示5-FU/HA佐劑治療的效用（6週的療 程）：在癌淋巴節之分散與新腫瘤形成上的效用。 第2 0圖顯示6個月效用研究之腫瘤一般外觀。 第2 1圖顯示5 - F U / Η A佐劑治療在病人存在上的效 用。 第22圖顯示在以CMF/HA治療達6週期間的小鼠在體 質上的變化％。 第23圖顯示在以CMF/HA治療達6週期間的小鼠在腫 瘤體積上的變化％。 第24圖顯示溶解於H20内的MTX之1H NMR光讀。 在光譜十分容易地鑑定出在ΜΤΧ中的氫原子群。 第25圖顯示ΜΤΧ與ΗΑ可能的相互作用。 第26圖顯示在600 MHz與298下透明質酸的6〇〇 MHz光譜。 -14- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） —ki-Kl· 卜'----— (請先閲讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（） 12 (請先閲讀背面之注意事項再填寫本頁) 第27圖顯示，在298 K下，僅有MTX與MTX及增加 加入有2毫微莫耳、1〇毫微莫耳、2〇毫微莫耳與80毫微 莫耳之 HA(50kDa)的 600 MHz WMR 光譜。 第28圖顯示，在298 K下，5-FU與5FU (1.25mg/mL、1.6mg/mL 與 6.4mg/mL 及 HA(750kDa，3mg/mL)在 7.0與 8.5ppm之間的 6〇〇 MHz 1NMR光譜。 縮窵 BSA 牛血清蛋白Figure 1 shows that at higher concentrations, HA forms a three-dimensional screen which is capable of entraining small molecules such as methotrexate. The HA/drug that locks the pathological location rapidly binds to the particular retention receptor via the HA, and then diffuses the drug from the HA and/or via both the HA and/or drug receptor A and the drug. Achieved by internalization. Figure 2A shows the possible molecular interactions between methotrexate and ha. This includes (i) ionic bonding, (ii) hydrogen bonding, or (iH) hydrophobic bonding. Figure 2B is a schematic diagram of the entanglement of a methotrexate in HA. At higher concentrations, HA forms a three-dimensional screen represented by large coiled molecules. (*) stands for methotrexate, which has only 454D and is very easily entrained in the 400-900 kD HA molecule. Figure 3 shows the general pathology of human breast cancer tumors in nude mice. Group A shows the morphology of human tumors of grade II_III. Panel B shows a micrograph of another section of the tumor shown in Figure 3A. The section shows that the surrounding mouse muscle (Μ), tumor sac (C), tumor necrotic area (Ν), infiltrating tumor (Τ), and (―) show a known as "Indian arrangement (Indian "Files" phenomenon in which the cancer cells are arranged in a column, which is generally associated with invasive cancer tumors (Carter, 1 990). Figure 4 shows the traditional pathological features of malignant tumors of the breast. Panel A shows a section of a feature of an invasive tumor with a large area of sudden death (N). This shows the tumor during the attack period. Group B shows the presence of blood cells (_ + ), which indicates that the tumor is attached to a blood vessel and thus survives. At a relatively close proximity to the vessel, there are some ducts (D) that can be observed. Group c shows that -12- This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) JI:------_衣|丨 (please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1280402 A7 B7 V. INSTRUCTIONS (10) Cells marked as (A), such as those displayed by nuclear fragmentation, suffer from apoptosis. Figure 5 shows the immunohistochemical identification of the Ha receptor on the fines of human breast cancer tumors grown in nude mice. Group A shows localization of carcinoembryonic antibodies (CF_A) in tumors. Panel B shows RHAMM performance on human breast cancer. Panel C shows the performance of IC A Μ -1 on human breast cancer. The localization of the ΗA receptor, ICAM-1 mainly surrounds the endothelial tissue (_>}. Group D shows the performance of 〇44 on human breast cancer. 0〇4411 is at about 9 5% It can be detected on tumor cells. Η: cells of human origin;] VI: cells of murine origin. Figure 6 shows the results of molecular weight analysis using hyalurance as a carrier for the locking of methotrexate for tumors. Methotrexate in human breast cancer tumors, using Η A as a carrier. Figure 8 shows enhanced uptake of methotrexate in the presence of HA. Figure 9 shows reduced intake of methotrexate in the gastrointestinal tract when the drug is administered with HA. The map shows a schematic representation of the possible tumor interaction between MTX/HA and its subsequent drug/Η A combination therapy. Figure 11 shows the cytotoxicity of the combined HA and 5FU in vitro. Figure 12 shows the use of HA as a carrier, 5-FU to lock human breast cancer tumors. -13- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) JI:-----Mine II ( Please read the note on the back first. Matters fill out this page) Order Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 A7 B7 V. Invention Description (11) Figure 13 shows the elimination path of HA in humans. Figure 14 shows the stomach, brain and lungs. Increased uptake of 5 _ FU. Figure 15 shows the effect of ha on plasma 5-FU pharmacokinetic elimination. Figure 16 shows the criteria used to define the endpoint of the experiment. It shows standard 2 (Group A) and Standard 3 (Group b). Figure 17 shows the utility of 5-FU/HA adjuvant treatment (6-week course): utility on primary tumor volume. Figure 18 shows 5-FU/HA adjuvant treatment The utility (6 weeks of treatment) · the effect on the main body weight. Figure 19 shows the effect of 5-FU / HA adjuvant treatment (6 weeks of treatment): in the dispersion of cancer lymph nodes and new tumor formation Fig. 20 shows the general appearance of the tumor in the 6-month utility study. Figure 21 shows the effect of 5-FU/ΗA adjuvant treatment in the presence of the patient. Figure 22 shows the treatment in CMF/HA up to 6 % change in body size of mice during the week. Figure 23 shows mice treated with CMF/HA for 6 weeks % change in tumor volume. Figure 24 shows 1H NMR optical reading of MTX dissolved in H20. The hydrogen atom group in the ruthenium is easily identified in the spectrum. Figure 25 shows the possible interaction of ruthenium and osmium. Figure 26 shows the 6〇〇MHz spectrum of hyaluronic acid at 600 MHz and 298. -14- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) —ki-Kl· Bu'--- - (Please read the note on the back and fill out this page) - Booked by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed 1284042 A7 B7 V. Invention Description () 12 (Please read the note on the back and fill out this page) Figure 27 shows that at 298 K, only MTX and MTX are added with an addition of 2 nanomoles, 1 nanomole, 2 nanomole, and 80 nanomoles of HA (50 kDa). 600 MHz WMR spectrum. Figure 28 shows that at 298 K, 5-FU and 5FU (1.25mg/mL, 1.6mg/mL and 6.4mg/mL and HA (750kDa, 3mg/mL) are between 7.0 and 8.5ppm. MHz 1 NMR spectrum. Bismuth BSA bovine serum albumin

Ci 居里 CMF 環磷醯胺，氨甲喋呤與5 -氟尿嘧啶 C y c 環磷醯胺 DNA 去氧核醣核酸Ci Curie CMF cyclophosphamide, methotrexate and 5-fluorouracil C y c cyclophosphamide DNA deoxyribonucleic acid

Dpm 每分鐘的惡化情況 DTTP 去氧胸腺嘧啶三磷酸 ECM 細胞外母質 EDTA 二乙撐基二胺四乙酸 經濟部智慧財產局員工消費合作社印製 ELISA 酵素鏈結之免疫吸附分析 F C S 胎牛血清 5-FU 5 -氣尿哺ϋ定 GAG 糖胺聚醣Dpm Deterioration per minute DTTP Deoxythymidine ECM Extracellular matrix EDTA Diethylenediaminetetraacetic acid Ministry of Economic Intelligence Intellectual Property Agency Employees Consumption Cooperative Printed ELISA Enzyme Link Immunosorbent Assay FCS Fetal Bovine Serum 5 -FU 5 - gas urinary feeding to determine GAG glycosaminoglycan

GlcNAc N -乙醯基胺葡糠胺 G1 c U A 葡糖酸酸 HA 透明質/透明質酸 -15-GlcNAc N - acetylamine glucosamine G1 c U A gluconic acid HA hyalin / hyaluronic acid -15-

1284042 A7 B7 五、發明説明（） 13 HABP 透明質結合蛋白 HAase 透明質酸酶 (請先閱讀背面之注意事項再填寫本頁) HBSS Hanks平衡鹽溶液 H RP 馬自由基過氧化酶 h 小時1284042 A7 B7 V. INSTRUCTIONS () 13 HABP hyaluronan binding protein HAase hyaluronidase (please read the back note and fill out this page) HBSS Hanks balanced salt solution H RP horse free radical peroxidase h hour

Kav 在凝膠内所獲致之體積分佈情況 1 升 min 分鐘 PBS 磷酸緩衝鹽溶液 PM 血漿膜 RHAMM 調節HA-移動性之受體 RMCA 大鼠之鼠類癌症 RNA 核糖核酸 RT 室溫 S -相 合成相 S.D. 標準偏差 SEM 平均值的標準誤差 經濟部智慧財產局員工消費合作社印製The volume distribution of Kav in the gel 1 liter min min PBS phosphate buffered saline PM plasma membrane RHAMM modulates HA-mobility receptor RMCA rat rodent cancer RNA ribonucleic acid RT room temperature S-phase synthesis phase SD Standard Deviation SEM Average Standard Error Printed by Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative

Ve 溶析體積 ' 空隙體積Ve lysis volume 'void volume

Vt 總體積 TGD 腫瘤生長遲滯作用 TDT 腫瘤雙Ti 發明的詳細說明 -16- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1284042 A7 ----------B7 __ 五、發明説明（） 14 本發明將參見下列非限制性的實施例而進一步的說 明。但可以了解到的是，下列的實施例僅是例示說明，且 不可以任何方式而被視為對本發明上述揭示之一般性作限 制。特別是，雖然本發明就有關癌症方面作詳細的說明， 但可以了解的是，在此所發現的事實並不限於治療癌症。 例如，細胞毒性劑亦可使用於治療其它的病況；氨甲嗓吟 廣泛地被使用於嚴重的類風濕性關節炎。 ——致在裸鼠中人類乳癌膣痼的有效性與飧在齓癌艚 瘼上透明質受饉之廉位鐘定 為了建立出人類乳癌的合適動物模式，必須要進行病 理測試。對一要在生理中存活的腫瘤而言，新生血管化是 必要的，因此，微血管網可供應營養至該腫瘤上。血管化 的存在、導管的入侵、壞死、程式化死亡、高有絲***指 數與核不正常係為乳癌的所有特性。 人類乳癌細胞株 MDA-MB-468 (American Tissue Culture Collection，Rockville，U.S.A)係基於其對 HA受體、CD44、RHAMM與ICAM-1的表現而被選 出。該等細胞一般係在1 7 0 c m2的培養瓶中以單層形式生 長並被次培養於被添加有1 0 %胎牛血清（F C S )與1 0 # g/ml慶大黴素之Leibovitz L-15培養基。為了要注射 入小鼠，細胞係被生長成100%匯流（confluency)，在 0 · 0 5 %騰蛋白酶/ 〇 · 〇 1 〇/〇 E D T A溶液中作胰蛋白酶分 析，在Beckman TJ-6實驗台型離心機（Beckman， Australia)以400gav離心10分鐘而清洗二次，使用 -17- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） " ---II (請先閲讀背面之注意事項再填寫本頁) —訂' 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7_ 五、發明説明（） 15 一 Model-ZM Coulter計數器（Coulter Electronics • I '1 I I I I (請先閲讀背面之注意事項再填寫本頁}Vt total volume TGD tumor growth retardation TDT tumor double Ti invention detailed description-16- This paper scale applies Chinese national standard (CNS) A4 specification (210X297 mm) 1284042 A7 ----------B7 __ V. INSTRUCTIONS (14) The invention will be further described with reference to the following non-limiting examples. However, it is to be understood that the following examples are merely illustrative and are not to be construed as limiting the general scope of the invention disclosed herein. In particular, although the invention has been described in detail with respect to cancer, it will be appreciated that the facts discovered herein are not limited to the treatment of cancer. For example, cytotoxic agents can also be used to treat other conditions; methotrexate is widely used in severe rheumatoid arthritis. - The effectiveness of human breast cancer in nude mice and the insufficiency of hyalin in the cancer of the sputum. In order to establish a suitable animal model for human breast cancer, a pathological test must be carried out. For a tumor to survive in physiology, neovascularization is necessary, and therefore, the microvascular network can supply nutrients to the tumor. The presence of vascularization, catheter invasion, necrosis, stylized death, high mitotic index and nuclear abnormality are all characteristics of breast cancer. The human breast cancer cell line MDA-MB-468 (American Tissue Culture Collection, Rockville, U.S.A.) was selected based on its expression for HA receptor, CD44, RHAMM and ICAM-1. The cells are typically grown in a single layer in a 1700 cm 2 flask and subcultured in Leibovitz supplemented with 10% fetal bovine serum (FCS) and 10 #g/ml gentamicin. L-15 medium. In order to be injected into mice, the cell line was grown to 100% confluency and trypsin analysis in 0. 55% transcriptase/〇·〇1 〇/〇EDTA solution on the Beckman TJ-6 test bench Centrifuge (Beckman, Australia) is washed twice at 400gav for 10 minutes, using -17- paper scale for Chinese National Standard (CNS) A4 size (210X297 mm) " ---II (please read the back first) Note: Please fill out this page) - Order 'Ministry of Economics Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 A7 B7_ V. Invention Description () 15 A Model-ZM Coulter Counter (Coulter Electronics • I '1 IIII (please read the back first) Note on the page again}

England)來計數，並以1χ1〇8細胞/ ml再懸浮於不具有 血清之Leibovitz L-15培養液。 85隻不具胸腺的Balb/c/WEHI雌性裸鼠（Walter and Eliza Hall institute of Medical Research, Melbourne，Australia)，6至8週大，係被維持在特殊 不具有病原的條件下，並具有隨意取得之無菌的食物與 水。如上所述，準備千萬個M D A - Μ B - 4 6 8細胞，且直接 注射進入在各小鼠乳頭下的脂肪墊。在9 〇 %的小氣中可 觀察到腫瘤生長。當腫瘤生長是可以目視檢測到時，該腫 瘤的發展係以每週測量該腫瘤體積來監測。腫瘤體積係使 用下列程式而由三個垂直直徑來計算出的； V= (1/6) ρ (άχά2ά3) 其中·· V係為腫瘤體積（m m3)， t為腫瘤之第一直徑（mm)， L為腫瘤之第二直徑（mm)，以及 d3為腫瘤之第三直徑（min) (Lamszus et al， 經濟部智慧財產局員工消費合作社印製 1 997). 腫瘤接種後8週，該腫瘤平均尺寸為482·2 mm3 (seM， 3 9.8mm3)。 在導入腫瘤後約8週，2隻帶有腫瘤的小鼠係給予致 死劑量的戊巴比妥鈉（Nembutal)。在3分鐘内殺死該等 J、鼠’腫瘤係以手術取出並馬上固定於1 〇 %經緩衝之福 馬林達1 2小時。該經固定的腫瘤係在一連串的7 〇 _ 1 〇 〇 % -18- 冢紙張尺度適用中國國家標準（CNS ) M規格（21〇><297公董） ^---- 1284042 A7 B7 五、發明説明（） 16 酒精中脫水，而後以石臘包埋，再由其中切出2 - 4 /z m的 切片。該切片係被置於載玻片上、去臘並與水接觸。該載 玻片係在磷酸-緩衝的鹽溶液（PBS)中清洗3x5分鐘。利 用以1 0 %胎牛血清1 0分鐘而阻礙親異質蛋白，而後以 PBS浸洗。在室溫（RT)下應用檢測用的抗體達60分鐘。 該抗血清或抗體係為對抗RHAMM (Applied Bioligands Corporation (Manitoba, Canada))、 ICAM-1、CD44v6、CD44vlO、總 CD44H 與 and CAE者。所有其它的檢測抗體係購自於Zy me d (California，U.S.A)。該玻片係在PBS中清洗3x5分 鐘，且内源性過氧化酶活性係利用將之浸於〇.3%H202/ 甲醇中達20分鐘而被阻礙。在另一次的PBS清洗後，在 RT下應用過氧化酶-綴合之豬抗-免子第二抗血清 (Dako，Denmark)達60分鐘，而後在PBS中清洗3x5分 鐘。依據製造商指示製備Sigma快速DAB (3，3，-二胺 聯苯胺，Sigma，St. Louis，U.S.A)錠劑，並將 DAB 溶液在室溫下應用達5 -1 0分鐘。該載玻片以自來水清洗 1〇分鐘，以蘇木精作對應染色，脫水並放置。 蘇木精與伊紅-染色的乳癌腫瘤切片的檢視顯示所有 與腫瘤存活有關的一般特性，如第3與4圖所示者，此確 認了動物寄主成功地維持第II級人類乳癌。其具有幾個為 惡性腫瘤特性之特徵。該標示為（B)之載玻片上的切片顯 示出該等特性。在惡性腫瘤所觀察到的病理特性係在切片 (B )中，即 -19- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製England) was counted and resuspended in Leibovitz L-15 medium without serum at 1χ1〇8 cells/ml. 85 Balb/c/WEHI female nude mice without thymus (Walter and Eliza Hall institute of Medical Research, Melbourne, Australia), 6 to 8 weeks old, were maintained under special conditions without pathogens and were obtained at will. Sterile food and water. As described above, ten million M D A - Μ B - 4 6 8 cells were prepared and directly injected into the fat pad under the nipple of each mouse. Tumor growth was observed in 9 〇 % of the small gas. When tumor growth is visually detectable, the development of this tumor is monitored by measuring the tumor volume weekly. Tumor volume is calculated from three vertical diameters using the following formula; V = (1/6) ρ (άχά2ά3) where · · V is the tumor volume (m m3), t is the first diameter of the tumor (mm L, the second diameter of the tumor (mm), and d3 is the third diameter of the tumor (min) (Lamszus et al, printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative 1 997). 8 weeks after tumor inoculation, The average tumor size was 482·2 mm3 (seM, 3 9.8 mm3). About 8 weeks after the introduction of the tumor, 2 tumor-bearing mice were given a lethal dose of sodium pentobarbital (Nembutal). The J, murine tumor lines were sacrificed within 3 minutes to be surgically removed and immediately fixed in 1 〇 % buffered Fmarinda for 12 hours. The fixed tumor line is applied to a series of 7 〇 1 1 〇〇% -18- 冢 paper scales for the Chinese National Standard (CNS) M specification (21〇><297 dongdong) ^---- 1284042 A7 B7 V. INSTRUCTIONS () 16 Dehydrated in alcohol, then embedded in paraffin, and cut into 2 - 4 /zm sections. The section was placed on a glass slide, dewaxed and brought into contact with water. The slides were washed in phosphate-buffered saline (PBS) for 3 x 5 minutes. The pro-isoprotein was blocked with 10% fetal bovine serum for 10 minutes and then washed with PBS. The antibody for detection was applied at room temperature (RT) for 60 minutes. The antiserum or anti-system is against RHAMM (Applied Bioligands Corporation (Manitoba, Canada)), ICAM-1, CD44v6, CD44vlO, total CD44H and and CAE. All other detection resistance systems were purchased from Zy me d (California, U.S.A). The slides were washed in PBS for 3 x 5 minutes and endogenous peroxidase activity was blocked by immersing them in 3%.3% H202/methanol for 20 minutes. After another PBS wash, peroxidase-conjugated porcine anti-free secondary antiserum (Dako, Denmark) was applied at RT for 60 minutes and then washed in PBS for 3 x 5 minutes. A Sigma Fast DAB (3,3,-Diamine benzidine, Sigma, St. Louis, U.S.A.) lozenge was prepared according to the manufacturer's instructions and the DAB solution was applied at room temperature for 5 - 10 minutes. The slides were washed with tap water for 1 minute, stained with hematoxylin, dehydrated and placed. Examination of hematoxylin and eosin-stained breast cancer tumor sections revealed all the general characteristics associated with tumor survival, as shown in Figures 3 and 4, which confirmed that animal hosts successfully maintained Grade II human breast cancer. It has several characteristics that are characteristic of malignant tumors. The slices on the slide labeled (B) show these characteristics. The pathological characteristics observed in malignant tumors are in section (B), ie -19- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back and fill out this page) Printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative

1284042 A7 B7 五、發明説明（ 17 〇 高的核/細胞質比例 11) 有角度的核染色質與核仁 Hi)不規則的核膜 可獲致的結論為，第Π-ΪΠ級位準的腫瘤係能夠在裸 鼠的小鼠模式中被支持。第⑴⑴級位準的腫瘤一般有約 47% 的預後存活率（B1〇〇m and Richards〇n，1 957) 第11 -111級位準的腫瘤係具有下列特性： 0 中等核多形性、過高的核染色質與有絲***活 性，可觀察到腫瘤切面所顯示的所有特徵；以及 H) 很少或沒有導管形成 大區域的壞死（N)係與腫瘤分散的情況有相關的， 此示出一較為攻擊性的侵入期間（Carter，199〇)。該腫 瘤的浸潤邊緣係以（）顯示之。 本發明的主要目的係為有效性，即該在該等小鼠中所 建立之腫瘤的組織學上與細胞學上的行為係可與在天然人 類寄主中该等行為相比擬的。在達成此目的上，我們亦 顯示出，在小鼠中的腫瘤細胞係為人類來源，且可高度表 現出相關的HA受體，例如，rhAMM、CD44與推定 之IC AM-1。因為該腫瘤鎖定係可經由受體·調節之化部 人或結合作用而發生者係為一種假說，故必須要確認出 HA 受體、ICAM-1、CD44 與 RHAMM 的表現。第 5B- D圖顯示，該等受體全部係存在著。表丨列出在二種測試 瘤腫中該等受體表現的程度。 丨.h :^一 卜 ----- (請先閱讀背面之注意事項再填寫本頁) l·訂 經濟部智慧財產局員工消費合作社印製 -20- 1284042 A7 B7 五、發明説明（） 18 表1 :在人類乳臚瘤外移植物上透明質受饉的焱現 在腫瘤上的抗原決定基表現的百分比之比率指數係被量化成 0%1284042 A7 B7 V. INSTRUCTIONS (17 〇 high nuclear/cytoplasmic ratio 11) Angled nuclear chromatin and nucleolus Hi) Irregular nuclear membrane can be concluded as a Π-ΪΠ level tumor system Can be supported in mouse mode in nude mice. Tumors at level (1) (1) generally have a prognosis of approximately 47% (B1〇〇m and Richards〇n, 1 957). Tumor lines at the 11th-111 level have the following characteristics: 0 medium nuclear polymorphism, Excessive nuclear chromatin and mitotic activity, all features shown by the tumor section can be observed; and H) necrosis (N) with little or no duct formation in large areas is associated with tumor dispersion, this shows A more aggressive intrusion period (Carter, 199 〇). The infiltrating edge of the tumor is indicated by (). The primary object of the present invention is to be effective in that the histological and cytological behavior of the tumor established in such mice can be compared to such behavior in natural human hosts. To achieve this, we have also shown that tumor cell lines in mice are of human origin and are highly relevant to the expression of HA receptors, e.g., rhAMM, CD44 and putative IC AM-1. Since the tumor-locking system can be a hypothesis through the receptor-regulated human or binding, it is necessary to confirm the expression of HA receptor, ICAM-1, CD44 and RHAMM. Figures 5B-D show that all of these receptors are present. Table 丨 lists the extent to which these receptors behave in the two test tumors.丨.h :^一卜----- (Please read the notes on the back and fill out this page) l·The Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives Print -20- 1284042 A7 B7 V. Inventions () 18 Table 1: The ratio of the percentage of epitopes on the tumor that is present on the human chymoma explants. The ratio of the percentage of epitopes on the tumor is quantified to 0%.

1 - 2 5 % 2 6 - 5 0 % 5 1 - 7 5 % 7 6 - 1 0 0 % 經濟部智慧財產局員工消費合作社印製 + + + + + + + HA受體 功能 腫瘤上的分佈情況 在腫瘤 上抗原 決定基 之表現〇/〇 CD44H 顯著地結合並内部 化HA之異構型 (Culty et al., 1 992) 在除了一些基質細 胞之外的細胞上的 表現 + + + + CD44v6 在腫‘中的角色未 知，但當使用作為 一預後因子。表現 愈高，則存活的機 率愈低 (Friedrichs et al., 1 995) +-- CD44v3 常在乳癌中過度表 現（Friedrichs et al” 1995) - RHAMM 轉形與腫瘤細則侵 入上是必要的 (Hall et al.5 1 9 9 5 ) 浸潤腫瘤細胞群， 其在環繞壞死區域 之細胞上具有高度 表現 + + + " ICAM-1 結合並内部化 HA，推定的代謝 受體（McCourt et al., 1 9 9 4) 在基質細胞上所存 在者 - CEA 表現惡性腫瘤細胞 上之胎兒抗原 (Haskell，1 9 9 0) 在所有腫瘤細胞中 存在著 + + + + ' -21- 1284042 A7 I —---— ________B7 五、發明説明（） 19 實倒」-逄明質蘖物组成物之盤備典法射 (請先閲讀背面之注意事項再填寫本頁) 在e貫了裸鼠模型的有用性之後，現在可將之用以測 试H A / Μ T X調劑的有效性。 ΜΤΧ的儲備溶液藉由溶解粉狀ΜΤΧ於〇.5%w/v碳酸 納（PH 9)中被製備，並利用〇.9〇/〇w/v 之達成 24· 5 mg/ml的濃度。該儲備溶液經由一 〇·22#㈤的遽器 被過濾，以確保在加入[3h]氨甲喋呤前係為無菌，並且 以一注射_等級的氣化鈉將之稀釋至一注射濃度。用於裸 鼠以及人類的MTX之藥學動力學比較數據已經被刊行 (Inaba et al，1 9 8 8)，而被應用於本研究的設計中，以 儘可此的模擬人類冶療的劑量。個別的注射根據個別的老 鼠體型大小被製備，以達到於5 〇 # 1中輸送丨5 m g / k g MTX的目標（相等於平均體重6〇公斤的〇42ing/kg的人 類治療的劑量；I n a b a e t a 1，1 9 8 8)。 經濟部智慧財產局員工消費合作社印製 乾燥的HA(model Mr 8·9χ105 kDa)被加入至 24.5mg/ml MTX儲備溶液之一部分中並且掁盪溶解隔 夜，以提供一21mg/ml之最終濃度。為確保無菌，加入 慶大徽素至50//g/ml的濃度，並且於4°C下培養隔夜。 在加入[3H]氨甲喋呤之後，該HA/ΜΤΧ儲備混合物以注 射等級之氣化鈉被稀釋成注射濃度。注射係個別地根據老 鼠體型大小被製備，以於50#1中輸送15mg/kg MTX與 12.5mg/kg HA。以如此量的HA注入體内，於試驗期間 飽和動力學將被觀察（Fraser et al，1983)。 -22- 本紙張尺度適用中國國家標準（CNS〉A4規格（210X 297公釐) ' --- 1284042 A7 B7 五、發明説明（） 20 為確保於氨甲喋呤/HA注射混合物製備期間，HA 維持其分子量’該注射溶液於一 Sephacry丨S- 1000大 小& 別凝膠（Pharmacia，Uppsala，Sweden)上以 1.6cm x 70cm的管柱明細事項，2πι1的樣品大小， 18ml/h的流速以及2ml的餾份大小被分析。第6圖顯示 HA於混合過程中維持其分子量。 4 0隻小鼠被隨機分為的二組。第I組僅接受μ τ X， 而第2組接受ΜΤΧ/ΗΑ組合物治療法。動物被個別地置 於一注射盒中，並且該注射經由尾部血管被投藥。於每 一注射中包含有氣標記的氨甲嗓吟（被注入的每分鐘的衰 變量之平均（dpm) 土標準差（Sem): 19，159，146 土 1，3 3 6,8 1 9)之 15mg/kg MTX ± I2.5mg/kg HA 被輸 送。小氣被個別地置於一柔軟的、非可浸濕的塑膠圍攔中 如此尿液可被收集。於注射之後30min、丨h、2h、4h或 8h，小鼠藉腹腔注射〇 lml戊巴比妥鈉（Glax〇, Australia Pty. Ltd.，Melbourne，Australia)被麻 醉，並且使用針頭以及注射筒自心臟或是大血管收集血液 。於血液收集之後，該動物藉由頸部錯位被殺死。 血液被輸送到EDT A塗覆的玻璃管中並且藉由於 14,000gav離心l〇min製備血漿。在以100 # 1的 30%v/v過氧化氫脫色並且加入3ml HiSafell scintillant之後，放射性於一 50/zl等分試樣被計數。 為克服化學化學發光與光致發光，視樣品來源該樣品於一 Wallac 1410召-計數器中重覆3、7或20 d期間被計數 -23- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） — κ-κικκ---—— (請先閲讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 _____B7__ 五、發明説明（） 21 2 min。於被計數之間的期間，樣品於室溫下被儲存在黑 暗中。所有的計算結果於一穩定的樣品被執行，於此所有 的化能與光能螢光均已被移除。 為了決定被注入MTX於血漿中的百分比，需要計算 出每一老鼠的總血漿體積（ml)，使用下述公式： 老鼠大小（g)x老鼠血液體積（〇〇7)血液的血漿部份 x (0.59) 被注入的Μ T X於血漿中百分比接著被計算： .命漿體積（ml) X dpm/m 1血漿X 1 〇 〇 被注入d p m總量 =%於血漿中被注入的MTX 。 為確保一正確數量的ΜΤχ被輸送至血管，該位於尾 部血管的注射處被解剖並且計量Μ τ X。被留存於注射位 置的ΜΤΧ注射之平均百分比為3·78% (Sem: 〇·57%)。 被輸送至jk管的MTX量（可用以分配至組織以及腫瘤的 MTX)如下被計算： ，輸送至於注射筒注射材料之留存於注射位 &的 中量的差 Dpm/mg 置的Dpm1 - 2 5 % 2 6 - 5 0 % 5 1 - 7 5 % 7 6 - 1 0 0 % Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed + + + + + + + Distribution of HA Receptor Functional Tumors The expression of the epitope on the tumor 〇/〇CD44H significantly binds and internalizes the isoform of HA (Culty et al., 1992) on the cells other than some stromal cells + + + + CD44v6 The role in swollen 'is unknown, but when used as a prognostic factor. The higher the performance, the lower the chance of survival (Friedrichs et al., 1 995) +-- CD44v3 is often overexpressed in breast cancer (Friedrichs et al) 1995) - RHAMM transformation and tumor detail invasion is necessary (Hall Et al. 5 1 9 9 5 ) Infiltrating tumor cell populations with high expression on cells surrounding the necrotic area + + + " ICAM-1 binding and internalizing HA, putative metabolic receptors (McCourt et al., 1 9 9 4) The presence of stromal cells - CEA shows fetal antigens on malignant cells (Haskell, 1 9 9 0) + + + + ' -21 - 1284042 A7 I -- in all tumor cells --- --- ________B7 V. Invention Description () 19 Really Down" - 逄 蘖 蘖 组成 组成 组成 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( It can now be used to test the effectiveness of HA / Μ TX modulators. A stock solution of hydrazine was prepared by dissolving powdery mash in 〇.5% w/v sodium carbonate (pH 9), and a concentration of 24. 5 mg/ml was achieved using 〇.9〇/〇w/v. The stock solution was filtered through a 〇·22# (5) sputum to ensure that it was sterile prior to the addition of [3h] methotrexate, and was diluted to an injection concentration with an injection of grade of sodium hydride. Pharmacokinetic comparison data for MTX in nude mice and humans has been published (Inaba et al, 198) and was used in the design of this study to mimic the dose of human therapy. Individual injections were prepared according to the size of individual mice to achieve the goal of delivering 丨5 mg / kg MTX in 5 〇 # 1 (equivalent to a human treatment dose of 〇42 ng/kg with an average body weight of 6 〇 kg; I nabaeta 1,1 9 8 8). Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, dried HA (model Mr 8·9χ105 kDa) was added to one of the 24.5 mg/ml MTX stock solutions and dissolved overnight to provide a final concentration of 21 mg/ml. To ensure sterility, Qingdasu was added to a concentration of 50//g/ml and incubated overnight at 4 °C. After the addition of [3H] methotrexate, the HA/ΜΤΧ stock mixture was diluted to an injection concentration with an injection grade of vaporized sodium. Injections were prepared individually according to the size of the old mouse to deliver 15 mg/kg MTX and 12.5 mg/kg HA in 50#1. With this amount of HA injected into the body, saturation kinetics will be observed during the test (Fraser et al, 1983). -22- This paper scale applies to Chinese national standards (CNS>A4 size (210X 297 mm)' --- 1284042 A7 B7 V. Inventive Note () 20 To ensure that HA maintains its molecular weight during the preparation of methotrexate/HA injection mixture 'The injection solution was on a Sephacry® S-1000 size & gel (Pharmacia, Uppsala, Sweden) with a 1.6 cm x 70 cm column detail, a sample size of 2πι1, a flow rate of 18 ml/h and a 2 ml distillation. The size of the fraction was analyzed. Figure 6 shows that HA maintains its molecular weight during mixing. 40 mice were randomly divided into two groups. Group I received only μ τ X, while Group 2 received ΜΤΧ / ΗΑ composition Therapy. Animals are individually placed in a syringe and the injection is administered via the tail vessels. Gas injections of methotrexate are included in each injection (average of decay per minute injected (dpm) Soil standard deviation (Sem): 19,159,146 soil 1,3 3 6,8 1 9) 15mg/kg MTX ± I2.5mg/kg HA is transported. The small gas is individually placed in a soft, non- This urine can be collected in the wettable plastic enclosure. After the injection 3 0 min, 丨h, 2h, 4h or 8h, mice were anesthetized by intraperitoneal injection of 〇lml sodium pentobarbital (Glax〇, Australia Pty. Ltd., Melbourne, Australia) and using a needle and syringe from the heart or Large blood vessels collect blood. After blood collection, the animal is killed by cervical dislocation. The blood is delivered to an EDT A coated glass tube and plasma is prepared by centrifugation at 14,000 gav. After decolorization of 30% v/v hydrogen peroxide and addition of 3 ml HiSafell scintillant, the radioactivity was counted in a 50/zl aliquot. To overcome chemiluminescence and photoluminescence, the sample was sampled from a sample of Wallac 1410 - The counter is counted for 3, 7 or 20 d. -23- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) — κ-κικκ---- (Please read the back of the note first) Matters fill out this page) - Booked by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 1240442 A7 _____B7__ V. Invention Description () 21 2 min. During the period between being counted, the samples were stored in the dark at room temperature. all The results of the calculations were performed on a stable sample where all of the chemiluminescence and the fluorescence of the light were removed. To determine the percentage of MTX injected into the plasma, the total plasma volume of each mouse was calculated (ml). ), using the following formula: Mouse size (g) x mouse blood volume (〇〇7) Plasma fraction of blood x (0.59) The percentage of injected Μ TX in plasma is then calculated: . X dpm/m 1 plasma X 1 〇〇 was injected into the total amount of dpm = % MTX injected into the plasma. To ensure that a correct number of tendons are delivered to the blood vessel, the injection site at the tail vessel is dissected and meters τ τ. The average percentage of sputum injections that were retained at the injection site was 3.78% (Sem: 〇·57%). The amount of MTX delivered to the jk tube (which can be used to distribute MTX to the tissue and tumor) is calculated as follows: Dpm delivered to the injection volume of the injection material and retained in the injection position &

MTX 的=(mg) χ - P 量 (Dpm) -24 - 本紙張尺度適用中國規格（2ι〇χ297公瘦) -—- 丨卜· K l· ----__ (請先閱讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（） 22 輸送至血管之MTX的數量從此之後將被稱作”注射劑量” 〇 為了正確的比較樣品族群以及標準化於器官與各腫瘤 大小的輕微差異，於身體器官與腫瘤以及體液中的ΜTX 濃度以％被注入劑量/組織克重來表示。 被留存於注射位置的ΜΤΧ注射百分比之平均值為 3· 78% (SEM: 0.5 7%)。為了標準化如此之變化，該於 腫瘤及組織中被發現的dpm百分比以一被注入的dpm減 去被發現留存於注射位置的dpm百分比被計算。此一用 3：於此後被認做是可獲得的d p m或是可獲得的氨甲嗓吟 。其結果被摘述於表2中。 ---—— (請先閱讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 -25- 本紙張尺度適用中國國家標準（CNS )八4規格（210X297公釐） 1284042 Δ7 Α7 Β7 五、發明說明（23 ) 經濟部智慧財產局員工消費合作社印製 , s s s} OO—COHN。妖^啉冢^^婼^染画。s S ^^^4⑨ ^^¥i#xlw<^^^«Q4a X1W鉍·染 ® ee^ksw 务檠鹌鉍肩眾^^啪衾伯崦林 * 言 13ss?s·®^ g* 0〇· 〇· O ψ^< ο s 〇 g· 卜 o ο <s o τΗ Ο o .•一 BHr ?|- ?l ll ?·Ι ll ?l ll +v s 9 ?ι ?•丨 ?ι ?l Q W XA Ό 〇· .工 o' .2. ο； 〇 ·〇 S • · · _, O 2 fS .二: g P. fS ο •o' Ό· * n o .+1 、0 rj »n 〇· S o VO . • jp s o. %n s .4J? 4Hr s ll ?•丨 S 1 ?•丨 5 y, ll I rj ll ?i ?ι ?•丨 ?•丨 〇〇 4s c\ C7N VO o s g s η s VO 1-<； 〇\ IS ：；t .* ·. · ·.〇 o .〇 •“ 〇 o Ό； Ο o '·说 S ο. o o s ν〇 55 g ― o ?, g rs JO rs^ 2 Hr m •ΛΟ ?i ?ι ?! ?! ?l ί\ ?l I I. 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Invention Description () 22 The number of MTX delivered to the blood vessels will be called "injection dose" from now on. For correct comparison The sample population is also normalized to the slight difference in organ and tumor size, and the ΜTX concentration in body organs and tumors as well as in body fluids is expressed as % injected dose/tissue weight. The average percentage of sputum injections retained at the injection site was 3.78% (SEM: 0.5 7%). To standardize such changes, the percentage of dpm found in tumors and tissues was calculated by subtracting the percentage of dpm from which the injected dpm was found to remain in the injection site. This use 3: This is considered to be the available d p m or the available methotrexate. The results are summarized in Table 2. ---- (Please read the note on the back and fill out this page) - Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives - 25 - This paper scale applies to China National Standard (CNS) 8 4 specifications (210X297 mm) 1284042 Δ7 Α7 Β7 V. Description of invention (23) Printed by the Consumers' Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs, ss} OO-COHN. Demon ^ 冢 冢 ^ ^ 婼 ^ dye painting. s S ^^^49 ^^¥i#xlw<^^^«Q4a X1W铋·染® ee^ksw 檠鹌铋 众 ^ ^^啪衾啪衾崦崦*言13ss?s·®^ g* 0 〇· 〇· O ψ^< ο s 〇g· 卜o ο <so τΗ Ο o .•一BHr ?|- ?l ll ?·Ι ll ?l ll +vs 9 ?ι ?•丨?ι ?l QW XA Ό 〇· .工o' .2. ο; 〇·〇S • · · _, O 2 fS . 2: g P. fS ο •o' Ό· * no .+1 , 0 rj » n 〇· S o VO . • jp s o. %ns .4J? 4Hr s ll ?•丨S 1 ?•丨5 y, ll I rj ll ?i ?ι ?•丨?•丨〇〇4s c\ C7N VO osgs η s VO 1-<; 〇\ IS :;t .* ·. · ·.〇o .〇•“ 〇o Ό; Ο o '·say S ο. oos ν〇55 g ― o ? , g rs JO rs^ 2 Hr m •ΛΟ ?i ?ι ?! ?! ?l ί\ ?l I I. 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This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm). 1284042 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumption Cooperative Printed A 7 B7 Explanatory note () 24 When the drug was co-injected with HA, no statistically significant difference was found in the plasma μTX level. The overall pharmacokinetics of sputum remained unchanged. After 5 to 2 hours of intravenous administration, a maximum sputum plasma level was reached (ΜIM S, 197). When possible urine is collected from the non-wettable plastic enclosure with a syringe and needle. The urine was clarified at 4, 〇〇〇gav centrifugation for 1 〇 min. The radioactive contents were measured after adding 3 ml of HiSafell scintillant to a sample of 8 - 30 v 1 . Although there is a technical difficulty in accurately quantifying the urine of each mouse, we calculate the percentage of the injected dose of T X in the urine by the following formula.

收集時間（h) X 42 "1 X dpm/“l 尿液 X 100 被注入總d p m =%於尿液中被注入的Μ T X 因為排尿速度的不同’自每一老鼠收集尿液是不可能 的。當每一時間點每一治療可獲得3個或更多的尿液樣本 時’於該等時間點每一數據無-母數統計分析被執行。於 投藥後一小時，有50% (ρ = 0.04 3)或是更多的ΜΤΧ於接 受ΜΤΧ/ΗΑ之小鼠的尿液中（見Table 2)。 在殺死老鼠之後立刻切下該腫瘤、肝臟、心臟、脾臟 、膀胱、左右腎臟、子宮、肺臟、胃、腸、腦以及淋巴結 並且整個的分析。於各組織中的放射性總量藉由溶解 1 00-400mg 組織於 3-6ml 的 OptiSolv (ACC, Melbourne，Australia)中歷時 36 h，22 °C 來被量測。 在溶液化完成後，於組織中的放射性在加入1 0 ml的 -27- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） ---續-------一訂—-----，|_,Ίί—小— (請先閲讀背面之注意事項再填寫本頁) 1284042 A7 B7 五、發明説明（） 25 (請先閱讀背面之注意事項再填寫本頁)Collection time (h) X 42 "1 X dpm/"l Urine X 100 is injected with total dpm =% in the urine is injected Μ TX because of the difference in urination speed 'It is impossible to collect urine from each mouse When 3 or more urine samples are available for each treatment at each time point, 'each data is not - the number of parental statistics is performed at these points in time. 50% after one hour of administration ( ρ = 0.04 3) or more in the urine of mice receiving sputum/sputum (see Table 2). Immediately after killing the mouse, the tumor, liver, heart, spleen, bladder, left and right kidneys were cut. , uterus, lungs, stomach, intestines, brain, and lymph nodes and the entire analysis. The total amount of radioactivity in each tissue was dissolved in 100-400 mg of tissue in 3-6 ml of OptiSolv (ACC, Melbourne, Australia) for 36 h. It is measured at 22 ° C. After the solution is completed, the radioactivity in the tissue is added to the 10 -27-sheet scale. The Chinese National Standard (CNS) A4 specification (210×297 mm) ---Continued -------One set------,|_,Ίί-small--(Please read the notes on the back first) Complete this page) 1284042 A7 B7 V. invention is described in () 25 (read the back of the precautions to fill out this page)

HiSafelll scintillant之後被計數。再一次的克服化學 發光與光致發光，樣品於Wallac 1410泠計數器中視樣 品來源重覆3、7或20 d期間。於計數之間的期間，樣 品於室溫下被儲存在黑暗中。所有的計算結果於一穩定的 樣品被執行，於此所有的化能與光能螢光均已被移除。圖 片表現平均土 SEM (n = 8)。 證實諸如肝臟、脾臟以及腎臟之代謝活動器官沒有表 現一高位準的藥物鎖定是很重要的，該鎖定會妨害任何增 加增加腫瘤鎖定的正向方面。表2例示了於不同時間點測 试氣甲嗓吟在每一組織的攝取。 利用 Mann-Whitney Rank Sum Test 以及 Students τ -test，當MTX與HA結合時，於MTX濃度 /組織克重並沒有統計學地顯著差異。由於在每一時間點 的小數量動物，若是數據重覆，Mann-Whitney test成 為統計學地無效的，但是於肝臟、子宮以及腸中，一明顯 的趨勢可被觀察到。 經濟部智慧財產局員工消費合作社印製 於肝臟中，當MTX與HA結合時，於Μτχ之攝取顯 示一短期的增加，如第7圖中所示者。 於3 Omin以及ih該肝臟含MTX濃度分別地平均增加 65 %以及26%。於2h時在與治療無關的肝臟中，Μτχ的 量沒有差異被觀察到。在4h之後，一令人感興趣的趨勢 變得很明顯，當MTX與HA共注射時，較少的“丁又於肝 臟中被發現（4h:少於68% MTX以及8h:少於75% MTX)’如第8圖所示。 -28- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇χ297公釐） 1284042 A7 B7 五、發明説明（ 26 經濟部智慧財產局員工消費合作社印製 於腸中有一顯著的趨勢，於此HA與MTX的組合物造 成藥物於每一時間點之攝取的減少，如第9圖所示。 腸被完全均質化，然後溶液化大約4〇〇rng的組織於 一 3-6ml的OptiSolv中於22°C下歷時24h，隨後加入 l〇ml的Hisafe-3 scintillant。為了克服化學發光與光 致發光，樣口口於一 Wallac 1410 /3-counter中視樣品來 源重覆3、7或2 0 d期間被計數歷時2 m i η。於計數之間 的期間，樣品於室溫下被儲存在黑暗中。所有的計算結 果於一穩定的樣品被執行，於此所有的化學與光致發光均 已被移除。 該等圖表現平均土 SEM (η = 8)。以無_母數隨機 測試分析該數據以配合展示ΗΑ之共投藥顯著地降低藥物 排入GI道中（p = 〇 · 〇 3 1，單尾測試）。 MTX濃度的減少範圍自43-67%。以無-母數隨機測 試分析該數據以配合展示HA之共投藥顯著地減少藥物排 入腸胃道中（p = 0 · 0 3 1，單尾測試）。 當與HA共投藥時，在4h時MTX顯著地較少出現於 肺臟中，以52%的平均減少（P = 〇〇14)。於其他時間、 沒有顯著的差異顯示’然而，此一觀察之該顯著性仍然 確定。 於脾臟、子宮、腦、心臟、淋巴結、胃以及腎臟中 有可觀察的趨勢被檢測出。 HA鎖定氨甲喋呤至腫瘤細胞的機制有兩種可能（第 1 0 圖）〇 點 不 沒 ------__ (請先閲讀背面之注意事項再填寫本頁) 訂HiSafelll scintillant is then counted. Once again, the chemiluminescence and photoluminescence were overcome and the samples were re-sampled in a Wallac 1410 泠 counter for 3, 7 or 20 d. During the period between counts, the samples were stored in the dark at room temperature. All calculations were performed on a stable sample where all of the chemical and light fluorescence had been removed. The image shows the mean soil SEM (n = 8). It is important to confirm that a metabolically active organ such as the liver, spleen, and kidney does not exhibit a high level of drug targeting, which can impair any positive aspects that increase tumor locking. Table 2 illustrates the uptake of air sputum in each tissue at different time points. Using Mann-Whitney Rank Sum Test and Students τ -test, there was no statistically significant difference in MTX concentration/tissue weight when MTX was combined with HA. Due to the small number of animals at each time point, the Mann-Whitney test became statistically ineffective if the data was repeated, but a clear trend was observed in the liver, uterus and intestine. The Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative is printed in the liver. When MTX is combined with HA, the intake of Μτχ shows a short-term increase, as shown in Figure 7. The liver containing MTX concentrations increased by an average of 65 % and 26%, respectively, at 3 Omin and ih. No difference in the amount of Μτχ was observed in the liver unrelated to treatment at 2 h. After 4h, an interesting trend became apparent. When MTX was co-injected with HA, less Ding was found in the liver (4h: less than 68% MTX and 8h: less than 75%) MTX)' is shown in Figure 8. -28- This paper scale applies to China National Standard (CNS) A4 specification (21〇χ297 mm) 1284042 A7 B7 V. Description of invention (26 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative There is a significant trend in the intestine, where the combination of HA and MTX causes a reduction in the intake of the drug at each time point, as shown in Figure 9. The intestine is completely homogenized and then solubilized by about 4 〇〇rng The tissue was plated in a 3-6 ml OptiSolv at 22 ° C for 24 h, followed by the addition of 1 ml of Hisafe-3 scintillant. To overcome chemiluminescence and photoluminescence, the mouth was viewed in a Wallac 1410 /3-counter The sample source was counted for 3, 7 or 20 d and was counted for 2 mi η. During the period between counts, the sample was stored in the dark at room temperature. All calculations were performed on a stable sample. All of this chemistry and photoluminescence have been removed. The average soil SEM (η = 8). The data was analyzed by random test without _ mother number to co-administer the drug to significantly reduce drug release into the GI tract (p = 〇·〇3 1, one-tail test). Concentrations ranged from 43-67%. Analysis of this data with a random-mother random test to demonstrate co-administration of HA significantly reduced drug release into the gastrointestinal tract (p = 0 · 0 3 1, one-tailed test). When co-administered with HA, MTX was significantly less present in the lungs at 4 h, with an average reduction of 52% (P = 〇〇 14). At other times, no significant difference was shown 'however, this observation should Significantness is still determined. Observable trends in the spleen, uterus, brain, heart, lymph nodes, stomach, and kidneys are detected. There are two possibilities for HA to lock methotrexate to tumor cells (Figure 10). No ------__ (please read the notes on the back and fill out this page)

-i - Bn HI -29- ‘紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1284042 A7 B7 五、發明説明（ 27 經濟部智慧財產局員工消費合作社印製 當Η A與MTX被結合時，有一顯著的鎖定效果（第7圖 )。藥物的留存於腫瘤中相對最大的增加為於〇.5h (平均 增加24%)、lh (平均增加30%)以及2h (平均增加 119%)，而於4h以及8h該增加係可忽略的。由於在每一 時間點的小數量動物以及數據的無母數*W，Mann-W h i t n e y R a n k S u m T e s t測試被使用，而當Η A被共注 射時，顯示於腫瘤中藥物攝取的顯著增加。於1 h時，該 統計學的顯著差異為ρ = 0·021，而於2h時ρ = 0·05。其他 的時間點沒有顯示一統計學地顯著差異，但是於ΜΤΧ濃 度/腫瘤克重的趨勢中，當與ΗΑ共注射時係一致的變大。 於第7圖中所展示的數據確定的顯示與ΗΑ共投藥於 注射後30min之内已經大大地增加腫瘤ΜΤΧ攝取。此外 ’於腫瘤中藥物濃度隨續的衰減中共投藥也維持了 一較高 的位準。於lh時ρ = 〇·〇21 (1^ = 8，n2 =8)，該差異是顯 著的，並且於2h時，當Ρ = 0· 05 (1^ =8，n2 =8)由無母 數測試決定。 包含夾帶有MTX之HA結合至該受體，（CD44及/或 I-CAM-1)並且經由一受體-調節的胞飲作用被内化，俾 以釋放該藥物至腫瘤細胞中。 ΗA分子篩網將會成為一向外擴散的障礙，如此於 HA結合至受體（CD 44、RHAMM及/或ICAM-1)之後 ’該被夾帶的Μ T X可以擴散至腫瘤細胞中。然而藉由η A 基質被留存於該細胞表面，MTX對於一般被利用以ΜΤχ 運送至細胞中主動運送具有增加可獲取性。 -30--i - Bn HI -29- 'The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1284042 A7 B7 V. Invention description (27 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Η A and MTX were When combined, there is a significant locking effect (Figure 7). The relative maximum increase in drug retention in the tumor is 〇5h (average increase of 24%), lh (average increase of 30%), and 2h (average increase of 119%) ), and the increase is negligible at 4h and 8h. Because of the small number of animals at each time point and the number of data without parent *W, the Mann-W hitney R ank S um T est test is used, and when When A was co-injected, it showed a significant increase in drug uptake in the tumor. At 1 h, the statistically significant difference was ρ = 0·021, and at 2 h ρ = 0·05. Other time points did not show A statistically significant difference, but in the trend of sputum concentration/tumor weight, it is consistently larger when co-injected with sputum. The data shown in Figure 7 is determined to be co-administered with sputum after injection. Increased tumor sputum intake within 30 minutes In addition, the concentration of the drug in the tumor continued to a higher level with the subsequent attenuation. At lh, ρ = 〇·〇21 (1^ = 8, n2 = 8), the difference is significant, and At 2h, when Ρ = 0· 05 (1^ = 8, n2 = 8) is determined by the no-parent test. HA containing the MTX is bound to the receptor, (CD44 and / or I-CAM-1) And is internalized via a receptor-regulated pinocytosis to release the drug into the tumor cells. The ΗA molecular sieve will become an obstacle to outward diffusion, thus binding to the receptor (CD 44, RHAMM and / or ICAM-1) 'The entrained Μ TX can diffuse into the tumor cells. However, by retaining the η A matrix on the cell surface, MTX is generally used to transport 至 to the cells for active transport. Acquisition. -30-

(請先閲讀背面之注意事項再填寫本頁) —訂 1284042 B7 五、發明説明（ 28 經濟部智慧財產局員工消費合作社印製 副作用以及藥物除了活性所欲位置外對組織的擴散， 也可以藉由其與HA結合而被影響。例如，藉由受體媒介 的細胞攝取，以及於肝臟（80-90%)、腎臟（10%)、脾 臟以及骨髓的代謝，最後兩個部位的一些小變異，Η A — 般不存在血管中。因為預期的HA劑量似乎增加藥物的肝 輸送至少一短暫時間。於3〇min及lh時肝臟顯示MTX濃 度分別平均增加65%以及26%。於2h時沒有顯著的差異 在MTX於肝臟中的量被發現，沒有治療。在4h之後，一 感興趣的趨勢變得很明顯，當與Η A共注射時，於此較少 MTX於肝臟中被發現（4h:少於68%的MTX以及8h:少於 7 5%的MTX)。在‘脈投藥之後，Μτχ於體液中被廣泛 的散布，並且可被留存於肝臟中達數個月（McEv〇y， 1988)，因此當於ha共注射時，MTX在肝臟於4h與8h 減少的平均濃度指出一藥學動力學清除路徑中改變的平衡 。慮及HA於肝臟内皮細胞（LEC)中快速的被代謝，接下 來Μ T X與Η A被於肝臟的竇狀線細胞中被共内化被釋放， 於此其可被分布至肝細胞中以分泌至膽汁中並且隨之進入 腸胃道，或是回到循環中以進一步的擴散進入體液中並且 隨尿液排出，或是同時。 短期肝部鎖定有一個冶療的優點。於肝臟轉移的情況 中，一快速的、高暴露至MTX可為有益的，並且所觀 到的鎖定僅有1 h，如此將中和任何長效毒性的問題。 臟鎖定可以與需要生物活性的藥物被利用，例如：絲 霉素C、亞德里亞黴素，於此該藥物/H a混合物將被鎖 察 肝裂 ---KtrK--F rrk--I __ , f請先閲讀背面之注意事項再填寫本頁j -訂 _ I I I · -31 - 1284042(Please read the precautions on the back and fill out this page) - Book 1284042 B7 V. Inventions (28 Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives print side effects and the spread of drugs in addition to the active position, can also borrow It is affected by its binding to HA. For example, cellular uptake by receptor mediators, and metabolism of the liver (80-90%), kidney (10%), spleen, and bone marrow, some small variations in the last two sites , Η A — does not exist in the blood vessels. Because the expected HA dose seems to increase the liver delivery of the drug for at least a short period of time. At 3 〇 min and lh, the liver showed an average increase of 65% and 26% in MTX concentrations. Significant differences were found in the amount of MTX in the liver, no treatment. After 4 h, a trend of interest became apparent, and when co-injected with ΗA, less MTX was found in the liver (4h) : less than 68% of MTX and 8h: less than 75% of MTX. After the administration of the vein, Μτχ is widely dispersed in the body fluid and can be retained in the liver for several months (McEv〇y, 1988), so when in ha At the time of injection, the mean concentration of MTX decreased in the liver at 4h and 8h indicates a balanced balance in the pharmacokinetic clearance pathway. Considering that HA is rapidly metabolized in liver endothelial cells (LEC), then Μ TX and Η A are It is released by co-internalization in the sinusoidal cells of the liver, where it can be distributed into hepatocytes for secretion into the bile and subsequently into the gastrointestinal tract, or back into the circulation for further diffusion into the body fluids. And with urine discharge, or at the same time. Short-term liver lock has the advantage of a cure. In the case of liver metastasis, a quick, high exposure to MTX can be beneficial, and the lock is only 1 h, this will neutralize any long-acting toxicity problem. Dirty locking can be utilized with drugs that require biological activity, such as: sermycin C, doxorubicin, where the drug/H a mixture will be locked Liver fissure---KtrK--F rrk--I __ , f Please read the notes on the back and fill out this page j-book _ III · -31 - 1284042

29 定至LEC。藉由於LEC中不活化藥物濃縮於LEC中，其 將可被擴政至肝細胞以活化，如此活化作為一活化鎖定機 制。 MTX的毒性的主要位置之一係為腸胃道。mtx與 Η A共投藥顯著地減少了輸送到腸胃道藥物的數量。有數 個機制與於腸胃道中Μ TX減少的濃度結合。 氰甲喋吟係為非常小的分子，其一般可被預期會通經 大部分的微血管壁，然而與ΗΑ結合會大大地將降低其通 經此路徑。 ΗΑ於肝臟内皮細胞的快速降解造成μτχ快速的釋放 進入肝臟並且回到血管中，於此其將經歷增加的腎臟的清 除’如於lh時所指出者，並且於接受mtx/ha調劑之小 鼠的尿液中多於50%的MTX被排出。 用以自身體中最後的消除MTX的主要方法是尿液的 排出。於尿液内容物中的藥物與Η A似乎有些許的增加， 但是我們相信由其他的證據顯示，ΗA可由腎臟快速的吸 收並且代謝’如此其存留時間會很短並且結合的藥物會很 快地釋放至尿液中。 結論 於此報導的結果顯示一用以檢測治療藥劑與透明質一 起投藥之前臨床試驗的第一階段，將可達成選擇性的輸送 所欲的病理組織標的；因此於該點達成一較高且較有效的 濃度。於此案例中，氨甲喋呤，一種廣泛的被用於現今治 療療法中以治療人類乳腺癌腫以及其他人類惡性疾病以及 n^i m» ---r.LTLKrt----- (請先聞讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 -32- 1284042 A7 B7 五、發明説明（ 30 類風滿性關節炎之一細胞毒性藥物，已經藉由與/不與透 明質投藥的靜脈注射路徑而被研究。 (請先閲讀背面之注意事項再填寫本頁) 我們已經發現裸鼠模型係為一特別適合用於研究於活 寄主中之人類乳癌腫瘤的模型，並且於使用Η A至直接治 療藥劑至罹病組織解決了基本的問題，同時因此加強了其 有益的效用。我們的結果已經顯示並未與Η A以特定形式 結的小分子藥物，當被注入血管時，其展現Η A仍然可以 輸送如此的藥物於一增強的量至該病理組織中。此一研究 顯示在體内佔優勢的條件，該Μ T X足以與Η A被留存以影 響輸送至人類乳癌的顯著增強，以及潛在的降低非所欲 的腸胃道毒性副作用。 里導U丄透明質糗物結合物之Μ僅以A涑射 證實裸鼠模型用於HA/佩里塔克爾的效用後，其可被 用於測試其他化學療法的效用。經確定，由於其在治療上 的重要性’佩里塔克爾（也被認為是紅豆杉醇）可以被使 用。 經濟部智慧財產局員工消費合作社印製 佩里塔克爾係由西方紫杉（Western Yew，raw 中被分離出來（Wani et al 1971)，且其臨 床上可有效地對抗卵巢癌及乳癌（Rownisky et al 1 990; McGuire et al 1 989)並且正被用於其他不同癌 症治療的試驗。然而，與佩里塔克爾結合主要的問題在於 其極端的親脂性以及伴隨極差的水溶性。解決這項問題的 努力已被引導至佩里塔克爾類似物及原藥的合成，以極度29 to LEC. By concentrating the inactive drug in the LEC in the LEC, it will be expanded to hepatocytes for activation, thus activated as an activation locking mechanism. One of the main locations of MTX toxicity is the gastrointestinal tract. Co-administration of mtx with ΗA significantly reduced the amount of drug delivered to the gastrointestinal tract. Several mechanisms are combined with a reduced concentration of Μ TX in the gastrointestinal tract. Cyanoguanidine is a very small molecule that is generally expected to pass through most of the microvascular wall, whereas binding to sputum will greatly reduce its passage through this path. Rapid degradation of liver endothelial cells causes a rapid release of muτχ into the liver and back into the blood vessels where it will undergo increased clearance of the kidneys as indicated at lh and in mice receiving mtx/ha modulators More than 50% of the MTX in the urine is discharged. The primary method used to eliminate MTX in the body is the discharge of urine. There appears to be a slight increase in the drug and ΗA in the urine content, but we believe that other evidence suggests that ΗA can be rapidly absorbed and metabolized by the kidneys so that its retention time will be short and the combined drugs will quickly Release into the urine. Conclusions The results of this report show that the first stage of a clinical trial to test the therapeutic agent together with the hyaluronan will achieve selective delivery of the desired pathological tissue; therefore, a higher and higher level is achieved at this point. Effective concentration. In this case, methotrexate, a widely used in today's therapeutic therapies to treat human breast cancer and other human malignancies, and n^im» ---r.LTLKrt----- (please read the back) Note: Please fill out this page again) - Booked by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives - 32- 1284042 A7 B7 V. Description of the Invention (A cytotoxic drug of 30 types of wind-filled arthritis, with/without The intravenous route of hyaluronic administration has been studied. (Please read the note on the back and fill out this page.) We have found that the nude mouse model is a model that is particularly suitable for studying human breast cancer tumors in living hosts, and The use of ΗA to direct therapeutic agents to rickets has solved the basic problems and thus enhanced its beneficial effects. Our results have shown that small molecule drugs that are not associated with ΗA in a specific form, when injected into blood vessels , which demonstrates that Η A can still deliver such a drug to the pathological tissue in an enhanced amount. This study shows that in the body predominating conditions, the Μ TX is sufficient to Η A Retention to affect the significant increase in delivery to human breast cancer, and potentially reduce the toxic side effects of unwanted gastrointestinal tracts. The 丄 丄 丄 丄 丄 丄 丄 丄 证实 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸 裸After the utility of Ritaker, it can be used to test the effectiveness of other chemotherapies. It has been determined that because of its therapeutic importance, Peritalake (also known as taxol) can be used. The Intellectual Property Bureau employee consumption cooperative printed the Peri Tucker is isolated from Western Yew (Wani et al 1971) and is clinically effective against ovarian and breast cancer (Rownisky et al 1) 990; McGuire et al 1 989) and is being used in trials for other different cancer treatments. However, the main problem associated with Perritakel is its extreme lipophilicity and its extremely poor water solubility. Efforts have been directed to the synthesis of Peritakiel analogues and original drugs to extreme

1284042 A7 B7 五、發明説明（） 31 的努力以創造出安全且生物可相容的調劑。截至目前沒有 原藥已經顯示了足夠的安定性、溶解度或是活性足以證明 其臨床的發展（Mathew et al 1 992; Vyas et al 1993)。然而，半合成的taxanes較佩里塔克爾顯示了 更大的溶解度以及潛力（Bissery et al 1991)並且其中 的一種形式Tax oters已經進入人體試驗階段（Bis set et al 1993) ° 現今被用於靜脈輸送的佩里塔克爾的臨床的配方使用 乙醇與Cremophor EL以一 1:1 (v/v)的比例與於6 mg/mL的藥物。Cremophor EL是確實的聚乙氧基的說 麻油；一清澄的、油性黏稠的、黃色的物質。安定性研究 已經顯示原始配方於4 °C下具有5年的櫃藏時間。該調劑 在使用前以0 · 9 %生理食鹽水或5 %右旋糖被稀釋至〇 · 3至 1.2 mg/mL的濃度並且該材料之物理的及化學的安定性 於此等條件下係為c a · 2 7 h。然而，載劑之稀釋至此等 位準可以產生一過飽和溶液（Adams et al 1993)若是 使用所指示的方法，如此傾向產生沉澱，於投藥時需要一 線濾器以確保避免注入粒子。同時也建議稀釋的 〇 a c 1 i t a X e 1溶液於製備後2 4小時内被使用。上述溶液的 霧狀物已經被觀察並且有助於排出of plasticisers by Cremophor 自該注入袋及管中（Waugh et al 1991) 〇 除了於物理的於先前所提及的安定性外， C r e m 〇 p h 〇 r最顯著的問題在於其經報導具有藥理活性之 事實。使用Cremophor EL數種不同的藥物被投藥，其 -34- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） _-hr rh--c. rrh-I---- , (請先閱讀背面之注意事項再填寫本頁) l·訂- 經濟部智慧財產局員工消費合作社印製 1284042 A7 _____B7 五、發明説明（） 32 (請先閱讀背面之注意事項再填寫本頁) 中包含環抱靈素、tacrolymus以及teniposide。然而， 被提供給佩里塔克爾的CremophorEL的劑量遠高於其 他任何標記藥物。Cremophoi:已經被觀察到會嚴重的或 不可過敏***物。當快速的注入佩里塔克爾動物或人類體 内時，載劑毒性也可能與不可避免的或致命的過敏反應被 觀察到（Dye & Watkins 1980; Lorenz et al 1977; Weiss et al 1 990)。 佩里塔克爾的儲備溶液係藉由個別地注射係根據個別 的老鼠的體重被製備。 經濟部智慧財產局員工消費合作社印製 乾燥的 HA(modal Mr 8.gxl〇5k Da)被加入至一 24.5mg/ml佩里塔克爾儲備溶液的部分中並且溶解掁盪 隔夜，以提供一 21mg/ml的最終濃度。為了確保無菌， 加入慶大黴素至一 50pg/ml的濃度並且於40°C下培養隔 夜。接下來加入[3H]佩里塔克爾，該HA/佩里塔克爾儲 備混合物以注射等級之氣化鈉被稀釋至注射濃度。注射係 個別地根據老鼠體形被製備，以於5〇 “I中輸送15mg/kg MTX與I2.5mg/kg HA。以如此量的HA注入體内，於 試驗期間飽和動力學將被觀察（F r a s e r e t a 1，1 9 8 3 )。 為了破保於佩里塔克爾/ H A注射混合物製備期間有 維持其分子量，該注射溶液於一 Sephacryl S-l〇〇〇大 小區別凝膠（Pharmacia，Uppsala，Sweden)以 1 Jcrn x 7〇cm的管柱詳細事項，2mi的樣品大小，流迷 18ml/h以及2ml餾分被分析。第4圖顯示HA於混合物製 備期間維持其分子量。 -35-1284042 A7 B7 V. INSTRUCTIONS (31) Efforts to create safe and biocompatible formulations. As of now, no original drug has shown sufficient stability, solubility or activity to justify its clinical development (Mathew et al 1 992; Vyas et al 1993). However, semi-synthetic taxanes show greater solubility and potential than Peritaker (Bissery et al 1991) and one of the forms, Tax oters, has entered the human trial phase (Bis set et al 1993). The clinical formulation of the delivered Perritaker uses ethanol and Cremophor EL in a ratio of 1:1 (v/v) to 6 mg/mL. Cremophor EL is a true polyethoxylated sesame oil; a clear, oily, viscous, yellow substance. Stability studies have shown that the original formulation has a shelf life of 5 years at 4 °C. The formulation is diluted to a concentration of 〇·3 to 1.2 mg/mL with 0. 9% physiological saline or 5% dextrose before use and the physical and chemical stability of the material is Ca · 2 7 h. However, dilution of the carrier to such a level produces a supersaturated solution (Adams et al 1993). If the indicated method is used, it tends to produce a precipitate which requires a line filter to ensure that the particles are not injected. It is also recommended that the diluted 〇 a c 1 i t a X e 1 solution be used within 24 hours of preparation. The mist of the above solution has been observed and contributes to the discharge of plasticisers by Cremophor from the injection bag and tube (Waugh et al 1991). In addition to the physical stability mentioned previously, C rem 〇ph The most significant problem with 〇r is the fact that it has been reported to be pharmacologically active. Several drugs of Cremophor EL are used for drug delivery. The -34- paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) _-hr rh--c. rrh-I---- , (please Read the precautions on the back and fill out this page. l·Settings - Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, Printing 1240442 A7 _____B7 V. Inventions () 32 (Please read the notes on the back and fill out this page) It is surrounded by linguistics, tacrolymus and teniposide. However, the dose of CremophorEL supplied to Perritak is much higher than any other labeled drug. Cremophoi: A severe or non-allergic insert has been observed. Carrier toxicity may also be observed with inevitable or fatal allergic reactions when rapidly injected into Perrithal animals or humans (Dye & Watkins 1980; Lorenz et al 1977; Weiss et al 1 990) . Peri Tucker's stock solutions were prepared by individual injections based on the weight of individual mice. The Ministry of Economic Affairs, the Intellectual Property Office, the employee consumption cooperative, printed dry HA (modal Mr 8.gxl〇5k Da) was added to a portion of a 24.5 mg/ml Perritak reserve solution and dissolved overnight to provide a 21 mg. The final concentration of /ml. To ensure sterility, gentamicin was added to a concentration of 50 pg/ml and incubated overnight at 40 °C. Next, [3H] Perritak, which was diluted to the injection concentration with an injection grade of sodium sulphate, was added. The injection system was prepared individually according to the shape of the mouse to deliver 15 mg/kg MTX and I2.5 mg/kg HA in 5" I. The amount of HA was injected into the body, and the saturation kinetics will be observed during the test (F Rasereta 1,1 9 8 3 ). In order to protect the molecular weight during the preparation of the Perritak/HA injection mixture, the injection solution was applied to a Sephacryl Sl〇〇〇 size differential gel (Pharmacia, Uppsala, Sweden). 1 Jcrn x 7〇cm column detail, 2mi sample size, flow fan 18ml/h and 2ml fraction were analyzed. Figure 4 shows that HA maintains its molecular weight during the preparation of the mixture.

1284042 A7 B7 五、發明説明（） 33 4 〇隻小鼠被隨機分為的二組。第I組僅接受佩里塔克 爾’而第2組接受佩里塔克爾/ η A組合物治療法。動物被 個別地置於一注射盒中，並且該注射經由尾部血管被投藥 。於每一注射中包含有氚標記的氨曱喋呤（被注入的每分 名里的衰變量之平均（dpm) 土 標準差（SEM): 19，159，146 土 1，3 3 6,8 1 9)之 1 5mg/kg MTX 土 1 2 · 5mg/kg Η A被輸送。小鼠被個別地置於一柔軟的、 非可次濕的塑膠圍棚中如此尿液可被收集。於注射之後 30min、lh、2h、4h或8h，小鼠藉腹腔注射01ml戊巴 比妥鈉（Glaxo，Australia Pty· Ltd., Melbourne, Australia)被麻醉，並且使用針頭以及注射筒自心臟或 是大血管收集血液。於血液收集之後，該動物藉由頸部錯 位被殺死。 血液被輸送到EDT A塗覆的玻璃管中並且藉由於 14,000gav離心10min製備血漿。在以1〇() " i 〇f 30%v/v過氧化氫脫色並且加入3mi msafeii scintillant之後，放射性於一 5〇//1等分試樣被計數。 為克服化學化學發光與光致發光，該樣品於一 WaUac 1 4 1 0冷-計數器中視樣品來源重覆於3、7或2 〇 d期間被 計數2 min。於被計數之間的期間，樣品於室溫下被儲存 在黑暗中。所有的計算結果於一穩定的樣品被執行，於此 所有的化能與光能螢光均已被移除。 為了決定被注入佩里塔克爾於血漿中的百分比，需要 计算出母老鼠的總血製體積（m 1 )，使用下述公式： --rrl· rr--rr rt.---__ , (請先閱讀背面之注意事項再填寫本頁) l·訂 經濟部智慧財產局員工消費合作社印製 -36-1284042 A7 B7 V. INSTRUCTIONS () 33 4 Two mice were randomly divided into two groups. Group I received only Peritaker's and Group 2 received the Perritak/η A composition treatment. The animals are individually placed in a syringe and the injection is administered via the tail vessels.氚-labeled ammonium is included in each injection (average of decay variables per minute (dpm) soil standard deviation (SEM): 19,159,146 soil 1,3 3 6,8 1 9) 1 5 mg/kg MTX Soil 1 2 · 5 mg/kg Η A is delivered. The mice were individually placed in a soft, non-wettable plastic enclosure so that urine could be collected. At 30 min, 1 h, 2 h, 4 h or 8 h after injection, mice were anesthetized by intraperitoneal injection of 01 ml sodium pentobarbital (Glaxo, Australia Pty. Ltd., Melbourne, Australia) and using a needle and syringe from the heart or Large blood vessels collect blood. After blood collection, the animal was killed by a cervical dislocation. Blood was delivered to EDT A coated glass tubes and plasma was prepared by centrifugation at 14,000 gav for 10 min. After decolorization with 1 〇 () " i 〇 f 30% v/v hydrogen peroxide and addition of 3 mi msafeii scintillant, radioactivity was counted in a 5 〇 / / 1 aliquot. To overcome chemiluminescence and photoluminescence, the sample was counted for 2 min in a WaUac 1 4 1 0 cold-counter depending on whether the sample source was repeated over 3, 7 or 2 〇 d. During the period between being counted, the sample was stored in the dark at room temperature. All calculations were performed on a stable sample where all of the chemical and light fluorescence had been removed. In order to determine the percentage of penitaxel injected into the plasma, the total blood volume (m 1 ) of the mother rat needs to be calculated using the following formula: --rrl· rr--rr rt.---__ , ( Please read the notes on the back and fill out this page. l·The Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printed -36-

A7A7

34 經濟部智慧財產局員工消費合作社印製 1284042 五、發明説明（） 老鼠大小（g)X老鼠血液體積（〇 〇7)血液的血漿部份 x (0.59) 被注入的佩里塔克爾於血漿中百分比接著被計算： 積（ml) X dpm/ml 血数 X 1〇〇 被注入dpm總量 ==%於血漿中被注入的佩里塔克爾。 為確保一正確數量的佩里塔克爾被輸送至血管，該位 於尾部血管的注射處被解剖並且計量佩里塔克爾。被留存 於注射位置的佩里塔克爾注射之平均百分比也被決定。被 輸送至血管的佩里塔克爾量（可用以分配至組織以及腫瘤 的佩里塔克爾）如下被計算： 被，送至 於注射筒 注射材料之 留存於注射位 血管的佩 中量的差 Dpm/m g 置的Dpm 里塔克爾—（mg) x - 的量 (Dpm) 輸送至血管之佩里塔克爾的數量從此之後將被稱作”注射 劑量”。 為了正確的比較樣品族群以及標準化於器官與各腫瘤 大小的輕微差異，於身體器官與腫瘤以及體液中的佩里塔 克爾濃度以％被注入劑量/組織克重來表示。 -37- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇χ297公董） (請先閲讀背面之注意事項再填寫本頁) ··衣· 訂 1284042 A7 B7 五、發明説明（） 35 留存於注射位置之佩里塔克爾注射的百分比之平均被 計算出來。為了標準化如此的變化，被發現於腫瘤及組織 中的dpm百分比以一注入dpm的百分比減去留存於注射 位置之dpm的百分比被計算。此一量被稱作可獲得的 dpm或可獲得的佩里塔克爾。 當可能的尿液由非可浸濕的塑膠圍欄以注射筒及針頭 被集後。尿液藉由於14,〇〇〇gav離心1〇 min被澄清。其 放射性的内容物於加入3 ml的HiSafell scintillant至 8 - 3 0 p 1樣品被計數。 在殺死老鼠之後立刻切下該腫瘤、肝臟、心臟、脾臟 、膀胱、左右腎臟、子宮、肺臟、胃、腸、腦以及淋巴結 並且整個的分析。於各組織中的放射性總量藉由溶解 1 00-400mg 組織於 3-6ml 的 OptiSolv (ACC， Melbourne, Australia)中歷時 3 6 h，22 °C 來被量測。 在溶液化完成後，於組織中的放射性在加入丨〇 m 1的 HiSafelll scintillant之後被計數。再一次的克服化學 發光與光致發光，樣品於Wall ac 1410 /3計數器中視樣 品來源重覆3、7或2 0 d期間。於計數之間的期間，樣 品於室溫下被儲存在黑暗中。所有的計算結果於一穩定的 樣品被執行，於此所有的化能與光能螢光均已被移除。 實例4 5_氟尿嘧啶糞HA之用途介紹34 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 V. Invention Description () Mouse Size (g) X Mouse Blood Volume (〇〇7) Plasma Part of Blood x (0.59) Infused Peritaker in Plasma The percentage is then calculated: product (ml) X dpm/ml blood number X 1 〇〇 is injected into the total amount of dpm ==% in the plasma is injected into Peritaker. To ensure that a correct number of Perritakels are delivered to the blood vessel, the injection site at the tail vessel is dissected and the Perritaker is metered. The average percentage of Perritak injections that were retained at the injection site was also determined. The amount of Peritucker delivered to the blood vessel (Peritacel that can be dispensed to the tissue and the tumor) is calculated as follows: The difference between the amount of the sputum and the amount of the sputum in the injection tube The amount of Dpm Ritakel-(mg) x - (mg) delivered to the blood vessel will be referred to as the "injection dose" thereafter. In order to correctly compare sample populations and normalize the slight differences in organ and tumor size, the Peritalkel concentration in body organs and tumors and body fluids is expressed in % injected dose/tissue weight. -37- This paper size is applicable to China National Standard (CNS) A4 specification (21〇χ297 公董) (Please read the note on the back and fill out this page) ····································· The average percentage of Perritak injections retained at the injection site was calculated. To standardize such changes, the percentage of dpm found in tumors and tissues was calculated as a percentage of a dpm injected minus the percentage of dpm retained at the injection site. This amount is referred to as the available dpm or the available Peritaker. When possible urine is collected from the non-wettable plastic fence with the syringe and needle. Urine was clarified by centrifugation at 14 〇〇〇gav for 14 〇 min. The radioactive content was counted by adding 3 ml of HiSafell scintillant to 8 - 30 p 1 sample. Immediately after killing the mice, the tumor, liver, heart, spleen, bladder, left and right kidneys, uterus, lungs, stomach, intestines, brain, and lymph nodes were cut and analyzed. The total amount of radioactivity in each tissue was measured by dissolving 100-400 mg of tissue in 3-6 ml of OptiSolv (ACC, Melbourne, Australia) for 3 6 h, 22 °C. After the completion of the solubilization, the radioactivity in the tissue was counted after the addition of HiSafelll scintillant of 丨〇 m 1 . Once again, the chemiluminescence and photoluminescence were overcome and the sample was repeated for 3, 7 or 20 d in the Wall ac 1410 /3 counter. During the period between counts, the samples were stored in the dark at room temperature. All calculations were performed on a stable sample where all of the chemical and light fluorescence had been removed. Example 4 Introduction of the use of 5_fluorouracil feces HA

5_氟尿嘧啶係為（5-FU)，一種抗代謝產物其經常被 用以治療乳癌及腸胃道癌（Piper & Fox，1982)。5-FU 細胞内地被轉換為其活化核苷酸形式，於此其同時干擾 -38- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） H.卜-' ri*·· 1 LJ· I* 1 11- - - I I (請先閱讀背面之注意事項再填寫本頁) —訂 經濟部智慧財產局員工消費合作社印製 1284042 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明説明（36 ) DN A與RN A合成。該藥物經由兩個活體中的機制被作用 (i) FdUmp緊密的結合至thymidylate合成酶以阻 礙thymidylate之形成，其係去氧三磷酸胸腺啶合成的必 要先趨物（DTTP)，其係用以合成DNA之四種去氧核糖 核苷酸之一。由此一阻礙所造成的缺乏胸腺嘧啶的狀態對 於活化地分化中細胞有毒性（Pinedo & Peters，1988) ο (ii) 5-FU作用的第二個路徑係為併合FUTP至RNA 中以干擾RNA作用。胸腺合成酶之阻礙由下述產生； Fdurd與5-FU併入至RNA中可以於細胞上產生細胞毒性 效果。暴露至5 - F U的濃度以及持續期間均為細胞毒性的 重要決定因素。因為針對RNA的效果於延長的暴露時間 中也許較佔優勢，因此針對DNA的效果在S相中短時間的 細胞暴露也許較為重要。 數項研究已經檢示使用Η A做為用於抗癌藥物的藥物 輸送的用途。（：〇^(1丨1^6{&1 ( 1 999)共價的結合丁酸鈉 至HA’以決定是否此一 ha/丁酸鈉組合物可增強於活體 中乳癌細胞的藥物攝取。此一研究指出該Η A做為一藥物 輸送载劑的機制可能涉及Η A- 丁酸鹽-酯衍生物被攜帶至 CD 44受體有關。該ha/藥物複合物被内化，隨之藉由 HA/藥物複合物的細胞質水解作用，接下來釋放出該丁酸 鹽其施行一抗增殖效果。一於活體中的研究被執行，其共 價地連結絲裂霉素C至HA作為一治療鼠性的路易士肺 木紙張尺度適用驗(—i )--~^. --rhrk--rrck---衣__ (請先閲讀背面之注意事項再填寫本頁) I訂 1284042 A7 B7 五、發明説明（37 ) 臟移植物的手段（Akima et al，1 996)。研究發現HA-MMC複合物於一 o.oimg/kg的劑量造成一抗-腫瘤活性 ’於此該單獨的藥劑，絲裂霉素C並未展現一抗-腫瘤活 性。 f例5於人類乳癌細胞中5-FU典HA之協同作用的讕杳 人類乳腺上皮癌細胞株M D A _ Μ B - 4 6 8、M D A - Μ B -43 5以及MDA-MB-231以與ΗΑ結合親合性（Culty et al，1 994)以及HA受體CD44以及RHAMM之表現 (Wang et-al，1 99 6)為基礎被篩選出來。該等細胞株之 特徵係如表3所述者。 表3:透明質結合以及人類乳胰上皮癌細胞株的受II裊现5_Fluorouracil is (5-FU), an antimetabolite that is often used to treat breast and gastrointestinal cancer (Piper & Fox, 1982). 5-FU cells are converted to their activated nucleotide form, which interferes with the -38- paper scale. It is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) H. Bu-' ri*·· 1 LJ· I* 1 11- - - II (Please read the note on the back and fill out this page) - Booked by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed 1284042, Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed A7 B7 DESCRIPTION OF THE INVENTION (36) DN A is synthesized with RN A. The drug is acted upon by two mechanisms in vivo (i) FdUmp binds tightly to thymidylate synthase to block the formation of thymidylate, which is a necessary precursor (DTTP) for thymidine synthesis of deoxytriphosphate. One of the four deoxyribonucleotides of synthetic DNA. The resulting thymidine-deficient state is toxic to cells in activated differentiation (Pinedo & Peters, 1988). (ii) The second pathway for 5-FU action is to combine FUTP into RNA to interfere RNA action. The inhibition of thymosin synthetase is produced by the incorporation of Fdurd and 5-FU into RNA to produce a cytotoxic effect on cells. Exposure to 5-F U concentrations and duration are important determinants of cytotoxicity. Since the effect on RNA may be dominant over extended exposure times, short-term cellular exposure in the S phase may be important for DNA effects. Several studies have examined the use of ΗA as a drug delivery for anticancer drugs. (: 〇^(1丨1^6{&1 (1 999) covalently binds sodium butyrate to HA' to determine whether this ha/sodium butyrate composition enhances drug uptake in breast cancer cells in vivo This study suggests that the mechanism by which Η A acts as a drug delivery vehicle may be related to the Η A-butyrate-ester derivative being carried to the CD 44 receptor. The ha/drug complex is internalized, followed by By the cytoplasmic hydrolysis of the HA/drug complex, the butyrate is subsequently released to exert an antiproliferative effect. A study in vivo is performed, covalently linking mitomycin C to HA as a The treatment of rat Luis lung wood paper scale test (-i)--~^. --rhrk--rrck---cloth__ (please read the note on the back and fill out this page) I book 1284042 A7 B7 V. INSTRUCTIONS (37) Means of Dirty Grafts (Akima et al, 1996). Studies have found that HA-MMC complexes cause a primary antibody-tumor activity at a dose of o.oimg/kg. The drug, mitomycin C did not exhibit primary antibody-tumor activity. f Example 5 in human breast cancer cells, 5-FU-type HA synergistic effect of human breast epithelium Cell lines MDA _ Μ B - 4 6 8 , MDA - Μ B -43 5 and MDA-MB-231 have binding affinity to ΗΑ (Culty et al, 994) and HA receptor CD44 and RHAMM (Wang Et-al, 1 99 6) was screened out. The characteristics of these cell lines are as described in Table 3. Table 3: Hyalin binding and human breast pancreatic epithelial cancer cell line

細胞株 乳癌型式 HA結合程度 HA受| 豐表現b CD44 RHAMM MD A-MB-23 1 + + + + + + + + MDA-MB- 468 + + + + + + + + + + MDA-MB- 435 + + + + ND a: Culty et al, 19 9 4 b: Wang et al，19 9 6 細胞株 MDA-MB-468、MDA-MB-435 與 MDA-MB-231如實例1所述者被例行的培養。 -40- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） --rl· t--rrrrk---^^^^衣__ (請先閲讀背面之注意事項再填寫本頁} —訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（38 ) 由表4中可以看見於含有〇nM 5-FU + lOOnm的培 養基中生長的乳癌細胞株，當與未治療細胞相較時展現一 統計學地顯著的增殖或細胞毒性的效果。 表:遑明質轸乳癌細胞增碴之故晃 細胞株 OnM 5-FU (平均細胞數± 1 S . D·) OnM 5-FU+100nM HA (平均細胞數± 1 s· D.) MDA-ΜΒ-23 1 0 = 4) 34,206 ±3,206 29,470 ± 4,452 — MDA-ΜΒ-468 (η = 4) 3 6,75 1 ±2,814 3 7,607 ±3,325 MDA-ΜΒ-43 5 (η = 4) 117,760 ±2,715 121,918 ±7,815 統計測試同時使用無_母數r a n k S u m T e s t以及母數 Student’s t-test 1^1. —ul·-»LrK n Hi In ^^1 ϋ (請先閲讀背面之注意事項再填寫本頁) I訂- 經濟部智慧財產局員工消費合作社印製 因此’所有的結果係一OnM 5-FU/0nm HA細胞計 量百分比被表現。雖然在細胞株之間生長模式的差異有被 注意到’例如MDA-MB-231及MDA-MB-468細胞株展 現了極差的平攤效果，於此無數的漂浮細胞於該測試培養 基之應用前存在。該MDA-MB-435細胞株展現一高平攤 效果及極高的生長速率。 -41-Cell line breast cancer type HA binding degree HA receiving | Feng performance b CD44 RHAMM MD A-MB-23 1 + + + + + + + + MDA-MB- 468 + + + + + + + + + + MDA-MB- 435 + + + + ND a: Culty et al, 19 9 4 b: Wang et al, 19 9 6 cell line MDA-MB-468, MDA-MB-435 and MDA-MB-231 as described in Example 1 Cultivation of the line. -40- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) --rl· t--rrrrk---^^^^衣__ (Please read the notes on the back and fill out this page. } —Subsidiary Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 A7 B7 V. Description of Invention (38) The breast cancer cell line grown in the medium containing 〇nM 5-FU + lOOnm can be seen in Table 4, when and not The treated cells showed a statistically significant effect on proliferation or cytotoxicity when compared with the cells. Table: OnM 5-FU (average cell count ± 1 S. D·) OnM 5-FU+100nM HA (average cell count ± 1 s·D.) MDA-ΜΒ-23 1 0 = 4) 34,206 ±3,206 29,470 ± 4,452 — MDA-ΜΒ-468 (η = 4) 3 6,75 1 ± 2,814 3 7,607 ±3,325 MDA-ΜΒ-43 5 (η = 4) 117,760 ±2,715 121,918 ±7,815 Statistical test using no _ parent number rank S um T est and parent number Student's t-test 1^1. —ul·- »LrK n Hi In ^^1 ϋ (Please read the notes on the back and fill out this page) I book - Ministry of Economic Affairs, Intellectual Property Bureau, employee consumption cooperative, printed A result-based OnM 5-FU / 0nm HA percentage of cells in an amount of performance. Although the difference in growth patterns between cell lines has been noted, 'for example, MDA-MB-231 and MDA-MB-468 cell lines exhibit extremely poor flattening effects, and the application of countless floating cells in the test medium is used. Pre-existing. The MDA-MB-435 cell line exhibited a high level of spreading effect and an extremely high growth rate. -41-

本紙張尺度適财@國家轉（CNS ) A4規格（21Qx297公瘦） 1284042 Δ7 Α7 Β7 五、發明説明（39 ) 當HA與5-FU被結合時，一協同的細胞毒性效果於 MDA-MB-468細胞株中被觀察到，但未出現於MDA-MB-231或MDA-MB-435乳癌細胞株中。此一結果顯示 於第11圖。5邛11單獨的1(：5()係>50卩111，但是當與11入結 合時IC5〇係為40nM，一於藥物劑量的降低高達1250倍 〇 當5 - F U於Η A存在下被應用於乳癌細胞時，於人工環 境下實驗的結果顯示一增加的細胞殺滅。該M D A - Μ B -468細胞對5FU/HA治療法顯示了最大的敏感性，於此 IC50 由〉50 " m減少至40nm，於此MDA-MB-231 與 MDA-MB-43 5並未非常地受到5-FU/HA組合物的影響 。Culty et al (1 994)所決定的，以 MDA-MB-468 (60-80%)、MDA-MB-231 (40-60%) g&MDA-MB-4 3 5 (4 0 - 6 0 % )，所有的乳癌細胞株表現C D 4 4受體的位 準。已經顯示於此實驗中所使用的3株細胞株可以降解 Η A，意指於腫瘤細胞中C D 4 4的作用可能在於調節η A的 降解（Culty et al，1 994)。 另一個考量的問題是細胞對化學療法的藥物之先前暴 露。在該細胞株由患者體内分離出來之前，癌症患者經常 會接受化學治療法或是放射線，其可造成腫瘤含有抗治療 細胞。於MDA-MB-435與MDA-MB-231的案例中，該 等細胞株由之分離的患者均已先被暴露至 (Cailleau et al，1 974)。由於癌症細胞含有數種適應 克服藥物細胞毒性的機制，在治療後留存的腫瘤質塊極有 -42- 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇Χ：297公釐） —rFtt---—— (請先閲讀背面之注意事項再填寫本頁) I訂 經濟部智慧財產局員工消費合作社印製 1284042 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明説明（4〇 ) 可能會對5 - F ϋ有抗性，接著於人工環境下改變該等細胞 對藥物的敏感性。 根據推測任何於活體中所取得的小鼠人類對應物治療 的數據的觀點’該試驗模型及設計係細心的被發展。為了 清顯示5-FU/HA佐劑治療法於人類乳癌外移植物於活體 中的鎖定以及療效，隨後的因子被檢視： 人類乳癌腫瘤於一經良好的-證實的Walter and Eliza Hall Institute品係裸鼠（一種廣泛的被接受的用 於如此之研究的模型）被證實，其避免了一對抗異源的細 胞免疫反應。 用於裸鼠及人類之5-FU之藥學動力學比較數據已經 被刊行（Inaba et al，1988)，並且被用於本研究之設計 ’以儘可能的模擬出人類治療上的劑量。 一主要的目標在於證實於此等小鼠中腫瘤組織學的以 及細胞學的狀態係與腫瘤於其自然的人類寄主中相類似的 。為達成這樣的目標，我們同時展示了位於人類來源中的 小鼠腫瘤細胞以及其等之表現與諸如RHAMM以及 CD44HA之HA受體高度相關。 資例6 HA及5_FU溶液之匍稱 一 HA的儲備溶液（ιοίβΜ，7mg/ml)經由溶解乾燥 的 HA (modal Mr 7x1 05 kDa)於 〇·9% w/v 無熱源注 射等級NaCl中被製備。為了確保溶液均質，該HAk4 °C溶解隔夜下然後充分振盪。為了確保H A於該儲備溶 ^ϋ· ^Lrl· BLri mi ϋϋ In —ϋ I (請先閱讀背面之注意事項再填寫本頁) 、1Τ -43- 五、 1284042 發明説明（41 ) 製備期間有維持其分子量，溶液於一 s -1 0 ο 0大小區別凝 膠中以1.6 cm X 70cm的管柱詳細事項、2ml的樣品大小 、1.81111/11的流速以及21]11餾分被分析。11八於一1〇〇111^ 的最終濃度，以所有的稀釋於適當的培養基中被製造。 5-FU儲備溶液藉由溶解5-FU於0.1M的氫氧化鈉並 以〇·9%\ν/ν之無熱源注射等級的NaCl使之成為 2〇mg/ml的濃度被製備。在加入（3H)_fu之前，該儲備 溶液經由一 0 · 2 2 # m的濾器被過濾以確保無菌，並且以 注射等級之氯化鈉稀釋至一注射濃度。個別的注射係根據 個別老鼠的體形大小被製備，以達成於5 〇 μ 1中輸送 30mg/kg 5-FU的目的（相等於人類治療上用於平均體重 60kg 之 l〇.5mg/kg 的劑量；inaba et al，1 98 8)· —無 熱源的HA 儲備溶液（l〇mg/ml; modal M，7x10 s Da) 被加入至20mg/ml 5-FU儲備溶液的一部分中並且掁盪 培養隔夜，至一相等於老鼠大小12.5mg/kg的最終HA 濃度。注射係根據老鼠大小個別地被製造，以5 0 v 1中輸This paper scale is suitable for the price @国转(CNS) A4 specification (21Qx297 瘦瘦) 1284042 Δ7 Α7 Β7 V. Description of invention (39) When HA and 5-FU are combined, a synergistic cytotoxic effect is on MDA-MB- It was observed in the 468 cell line, but not in the MDA-MB-231 or MDA-MB-435 breast cancer cell line. This result is shown in Figure 11. 5邛11 alone 1 (:5() system>50卩111, but when combined with 11-incorporation, the IC5 lanthanide is 40nM, and the drug dose is reduced by up to 1250 times. When 5-FU is in the presence of ΗA When applied to breast cancer cells, the results of experiments in an artificial environment showed an increased cell killing. The MDA-Μ B -468 cells showed the greatest sensitivity to 5FU/HA treatment, and the IC50 was >50 &quot m is reduced to 40 nm, where MDA-MB-231 and MDA-MB-43 5 are not very affected by the 5-FU/HA composition. Culty et al (1 994) determined by MDA-MB -468 (60-80%), MDA-MB-231 (40-60%) g&MDA-MB-4 3 5 (40-60%), all breast cancer cell lines exhibit CD 4 4 receptor The three cell lines used in this experiment have been shown to degrade Η A, meaning that the role of CD 4 4 in tumor cells may lie in the regulation of η A degradation (Culty et al, 1994). The problem to be considered is the previous exposure of the cell to the chemotherapy drug. Before the cell line is isolated from the patient, the cancer patient often receives chemotherapy or radiation, which can cause Tumors contain anti-therapeutic cells. In the case of MDA-MB-435 and MDA-MB-231, patients isolated from these cell lines were first exposed (Cailleau et al, 1 974). Several mechanisms for adapting to overcome the cytotoxicity of drugs, the tumor mass retained after treatment is extremely -42- This paper scale is applicable to China National Standard (CNS) A4 specification (21〇Χ: 297 mm)—rFtt---- — (Please read the precautions on the back and fill out this page.) I. Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Printing 1284042 Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, Printing A7 B7 V. Inventions (4〇) May be 5 - F ϋ is resistant, and then the sensitivity of the cells to the drug is changed in an artificial environment. According to the data of any mouse human counterpart treatment obtained in vivo, the experimental model and design are careful. In order to clearly show the locking and efficacy of 5-FU/HA adjuvant therapy in human breast cancer grafts in vivo, subsequent factors were examined: Human breast cancer tumors were once well-confirmed Walter an d Eliza Hall Institute strains of nude mice (a widely accepted model for such studies) have been shown to avoid a cellular immune response against heterologous. Pharmaceutics for 5-FU in nude mice and humans Kinetic comparison data has been published (Inaba et al, 1988) and used in the design of this study to simulate human therapeutic doses as much as possible. A primary goal was to demonstrate that tumor histology and cytological status in these mice are similar to tumors in their natural human hosts. To achieve this goal, we also demonstrated that mouse tumor cells located in human origin and their performance are highly correlated with HA receptors such as RHAMM and CD44HA. Example 6 A nickname of HA and 5_FU solution A stock solution of HA (ιοίβΜ, 7 mg/ml) was prepared via dissolved dry HA (modal Mr 7x1 05 kDa) in 〇·9% w/v non-pyrogenic injection grade NaCl. . In order to ensure homogenization of the solution, the HAk was dissolved at 4 ° C overnight and then shaken well. In order to ensure that HA is dissolved in the reserve, ^·^Lrl· BLri mi ϋϋ In —ϋ I (please read the note on the back and then fill out this page), 1Τ -43- 5, 1284042 Description of invention (41) Maintenance during preparation The molecular weight of the solution was analyzed in a s -1 0 ο 0 size difference gel with a 1.6 cm X 70 cm column detail, a 2 ml sample size, a 1.81111/11 flow rate, and a 21]11 fraction. The final concentration of 11 一 1 〇〇 111 ^ was prepared by diluting all of the appropriate medium. The 5-FU stock solution was prepared by dissolving 5-FU in 0.1 M sodium hydroxide and making it at a concentration of 2 〇 mg/ml in a non-pyrogenic injection grade of 〇·9%\ν/ν. Prior to the addition of (3H)_fu, the stock solution was filtered through a 0.22 m filter to ensure sterility and diluted to an injection concentration with an injection grade of sodium chloride. Individual injections were prepared according to the size of individual mice to achieve a delivery of 30 mg/kg 5-FU in 5 μμ 1 (equivalent to a dose of 0.5 mg/kg for human treatment for an average weight of 60 kg). ;inaba et al,1 98 8)·—Halogen-free HA stock solution (l〇mg/ml; modal M, 7x10 s Da) was added to a portion of the 20 mg/ml 5-FU stock solution and cultured overnight To a final HA concentration equal to 12.5 mg/kg in mouse size. The injection system is individually manufactured according to the size of the mouse, and is lost at 5 0 v 1

送 30mg/kg 5-FU 以及 12.5mg/kg HA。以如此量之 HA 被注射進入體内，飽和動力學於試驗期間將被觀察到 (Fraser* et al，1 983)。為了確保於該混合物的製備期間 HA維持其分子量，該注射溶液於一 sephacryl S-l〇〇〇 大小區別凝膠中，使用1 · 6 c m X 7 0 c m的管柱詳細事項 、2ml的樣品大小、1 8ml/h的流速以及2ml的餾分被分 析。於管柱餾份中的透明質藉由糖酸酸分析被檢測。 -44- 本紙張尺度適用中國國家標準（CNS ) A4規格（2丨〇'〆297公董） __rr t__r.LL_____ , (請先閱讀背面之注意事項再填寫本頁)Send 30 mg/kg 5-FU and 12.5 mg/kg HA. With this amount of HA injected into the body, saturation kinetics will be observed during the trial (Fraser* et al, 1 983). In order to ensure that the HA maintains its molecular weight during the preparation of the mixture, the injection solution is used in a sepikacryl Sl size differential gel, using a column of 1 · 6 cm X 70 cm, 2 ml sample size, 1 A flow rate of 8 ml/h and a fraction of 2 ml were analyzed. The hyaluronic acid in the column fraction was detected by analysis of the sugar acid. -44- This paper scale applies to China National Standard (CNS) A4 specification (2丨〇'〆297 公董) __rr t__r.LL_____, (please read the notes on the back and fill out this page)

、1T 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（42 ) (請先閲讀背面之注意事項再填寫本頁) 該糖.SiL为析自该荨由凝膠過渡色層分析法所收集的 顧份量化地被用以檢測透明質之存在。每一餾分2 5 # 1的 等分試樣接下來被轉移到9 6井穴盤中。2 5 0 // 1的咔唑類 試劑（3M carbazole/〇.〇25M 硼酸鹽於112804中）接 下來被加至該等餾分中。該96井穴盤於80°C被培養歷時 45_60min。一具有 550nm濾器的 Dynatech MR7000 plate reader被用來讀取該96井穴盤。當該吸光度>3標 準偏差高於背景吸光度時，其被視為顯著。該背景藉由於 Vo以及Vt之前與之後取得相同數目的樣品點該計算，於 此該取用的平均係為16 (Fraser et al. 1988)。 HA 與 5-FU 的配方（l〇mg/ml HA 與 20mg/ml 5-FU 於0.5% NaCl中，pH 8.9)並沒有顯示一於HA之分子 量上的降解效果。於大小區別凝膠中的色層分析法指出可 能的分子量為700,000Da。 實例7藥物細胞毒性分析：弒腌妫計 經濟部智慧財產局員工消費合作社印製 於人工環境下在設計藥物滴定實驗時，應用與在靜脈 注射投藥後血漿中可達藥物濃度相類似的濃度係為所欲者 。當5-FU藉由一靜脈注射路徑（iv)於10.5mg/kg (400mg /m2)被治療地被投藥，於30 min内血漿最位 準係為8/zg/ml [61/zM，.Kubo，1990]。以此等於活體 中的血漿濃度為基準，該等接隨的藥物濃度被選擇；〇 、1、5、10、20、50、100 // M 5_FU 土 ΙΟΟηΜ HA 。細胞株 MDA-MB-468、MDA-MB-231 以 MDA-MB-43 5如上述於實例4至5中特化者被培養。當該等細 -45- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1284042 A7 _____B7__ 五、發明説明（43 ) 胞達成匯流時，該等細胞自一於0 · 2 5 %胰蛋白/0 · 0 5 % EDTA的燒瓶中被移出。該單一 ·細胞懸浮液以一 Coulter計數器（ZMI model)被計數並且細胞重新懸浮 為1 00,0 00細胞/ml於細胞-特定培養基中。細胞被放入 24-井穴盤（2cm2表面積）藉由每井添加〇.5ml細胞懸浮 液，產生50,000細胞/井。 培養被容許持續24h，在培養基被移除之前，單一細 胞層以Η B S S清洗並且使用培養基（5 - F U + Η A)。在測試 培養基中生長4日後該培養以HB S S沖洗，該細胞以 0.2 5%胰蛋白/ 0.05% EDTA利用胰蛋白化自井中移出 並且以一 Coulter Counter計數細胞。 實例8 EA做為一薷物输送以及腫痼-麴定盤埘之砰仕 以實例5至7中人工環境試驗中藥物敏感度的結果， 以及HA受體CD44與RHAMM之表現為基礎，該人類乳 上皮癌細胞株M D A - Μ B - 4 6 8被選定做為該癌細胞接種源 用以產生任何裸鼠人類腫瘤外移植物。細胞被例行地培養 並且如貫例5中所述的次培養。為了注射至小鼠中，細胞 被培養至 100%匯流，於0.025%胰蛋白/0·01% EDTA溶液中騰蛋白化，藉由於Beckman TJ-6 bench centrifuge中在400g下離心lOmin清洗二次，利用一 Model-ZM Coulter counter被計數，並且以} χ 1〇8 細胞/ml重新懸於一無血清的Leibovitz L-15 medium 中ο -46- 本紙張尺度適用中國@家標準（CNS ) Α4規格（210x297公董)" ' ---Γ-hl· Llhc----__ Imp (請先閱讀背面之注意事項再填寫本頁) l·訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 發明説明（44 ) 該無胸腺之CBA/WEHI 6至8週大的雌性裸鼠，係 #皮、維持在特殊不具有病原的條件下，並具有隨意取得之無 菌的食物與水。每一老鼠接受一於50/zl中含有5 X 106 細胞的注射。該等細胞以一 2 6規範（g a u g e)的針頭被注 射至直接位於第一乳頭下方的乳腺脂肪墊中（Lamszus et al，1 99 7)。藉由量測三個與水平面呈直角的直徑，腫 瘤每週地被量測（did2d3)。腫瘤體積使用下列公式被估 計： (1 /6)71(1 d2d3) 使用5-FU ± HA之治療大約於癌細胞接種4至8週後開無 。被用於每一研究中該小鼠平均腫瘤大小摘述於表5中。 究開始時的人齄轧癌腫瘧捆述 腫瘤體積 (平均+ SEM) 腫瘤佔淨身體體積％ (平均土 SEM) 5 - F U銷定 〇·41 ±〇.〇lmm3 0.22 + 0.10mm3 有效期:6週 0 · 3 7 ± 0.2 0mm3 0.19 + 0.10mm3 有效期：6個Η 0.61 +0.36mm3 0.28 +0.17mm3 為證實當乳癌細胞株M D A - Μ B - 4 6 8被注射至一適' 的動物寄主中時之腫瘤產生力以及其產生腫瘤的能力，」 不需要進行一項病理學的測試。因為一腫瘤為病理地可」 存的jk管增生（新血管化）係為必需的，於此該微血管網彡 供給營養至該腫瘤。血管增生、導管的入侵、壞死、3 —_47· ^適用中國國家標準（CNS ) A4規格（21Qx297公楚） ' ' m· ^ri« ϋϋ —ϋ ϋ— ϋ (請先閲讀背面之注意事項再填寫本頁) 、1Τ 經濟部智慧財產局員工消費合作社印製 張 -紙 本 1284042 Α7 Β7 五、發明説明（45) 式化死亡、南有絲***指數與核不正常的出現均為乳上皮 癌的特徵。蘇木精與伊紅-染色的乳癌腫瘤切片的檢視顯 示所有與腫瘤存活有關的一般特性，如此確認了動物寄主 成功地維持第II級人類乳上皮癌。 在腫瘤誘生約8週之後，兩隻帶有腫瘤的小鼠被提供 一致死劑量的戊巴比妥鈉。於殺死小鼠後3分鐘内，腫瘤 被外科手術地移除並且立刻被固定於1 〇 %經緩衝的福馬 林中歷時2 h。該經固定的腫瘤係在一連串的7 〇 -1 〇 〇 〇/0酒 精中脫水，而後以石臘包埋，再由其中切出2-4/zm的切 片。該切片被置於一載玻片上，脫蠟並與水接觸。該載玻 片係在磷酸-緩衝的鹽溶液（PBS)中清洗3x5分鐘。利用 以10%胎牛血清10分鐘而阻礙親異質蛋白，而後以PBS 浸洗。在室溫（RT)下應用檢測用的抗體達6 〇分鐘。該抗 血清或抗體係為對抗RH AMM以及C AE。該玻片係在 P B S中清洗3 X 5分鐘，且内源性過氧化酶活性係利用將 之浸於0.3 % H2〇2 /甲醇中達20分鐘而被阻礙。在另一次 的PBS清洗後，在RT下應用過氧化酶-綴合之豬抗-免 子第二抗血清（Dako，Denmark)達60分鐘，而後在 PBS中清洗3x5分鐘。依據製造商指示製備sigma快速 DAB (3,3 -«^胺聯本胺，Sigma，St· Louis，U.S.A) 錠劑，並將DAB溶液在室溫下應用達54 〇分鐘。該載玻 片以自來水清洗1 0分鐘，以蘇木精作對應染色，脫水並 放置。 -48- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） __--ΓΓ t.----- (請先閱讀背面之注意事項再填寫本頁) 訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 —— _B7_ 五、發明説明（46) 腫瘤的人類來源經由以人類特定的癌症染色標記染色 該腫瘤及週圍組織而被確定。C E A的出現清楚的展現其 係為人類腫瘤，然而被維持於一鼠性的寄主的心血管系統 中。由於經由受體-調節内化或結合腫瘤鎖定係為一假說 ，證實HA受體、CD44與RHAMM之表現係為必要者。 表6列示於人類乳癌腫瘤外移植物受體表現的程度。 —-ΚΙ-ΙΓ hr hl· 111 1111 11 ^ (請先閲讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -49- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1284042 A7 B7 五、發明説明（47 ) 表^ 在於裸i中生長之人麴氪碑艙痼的分布 經濟部智慧財產局員工消費合作社印製 HA受體 功能 腫瘤上的分佈情況 在腫瘤 上抗原 決定基 之表現 % CD44H 顯著地結合並内 部化HA之異構 型（Culty et al·，1 992) 在除了一些基質細 胞之外的細胞上的 表現 + + + + C D 4 4 ν 6 在腫瘤中的角色 未知，但當使用 作為一預後因 子。表現愈高， 則存活的機率愈 低（Friedrichs et al·，1 995) + CD44v3 常在乳癌中過度 表現 (Friedrichs et al·，1 995) RHAMM 轉形與腫瘤細則 侵入上是必要的 (Hall et al., 1 995) 浸潤腫瘤細胞群， 其在環繞壞死區域 之細胞上具有高度 表現 + + + CEA 表現惡性腫瘤細 胞上之胎兒抗原 (Haskell， 1 990) 在所有腫瘤細胞中 存在著 + + + + 在腫瘤上的抗原決定基表現的百分比之比率指數係被量化成： 0% 1 - 2 5 % 2 6 - 5 0 % 5 1 - 7 5 % 7 6 - 1 0 0 % -50-1T Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1240442 A7 B7 V. Invention description (42) (Please read the back note first and then fill out this page) The sugar.SiL is from the gel transition layer The samples collected by the analytical method are quantitatively used to detect the presence of hyalin. An aliquot of each fraction 2 5 # 1 was then transferred to a 96 well well. The 2 5 0 // 1 carbazole reagent (3M carbazole/〇.〇25M borate in 112804) was then added to the fractions. The 96 well tray was cultured at 80 ° C for 45_60 min. A Dynatech MR7000 plate reader with a 550 nm filter was used to read the 96 well plate. When the absorbance > 3 standard deviation is higher than the background absorbance, it is regarded as significant. The background is calculated by taking the same number of sample points before and after Vo and Vt, and the average of the accesses is 16 (Fraser et al. 1988). The formulation of HA and 5-FU (l 〇 mg/ml HA and 20 mg/ml 5-FU in 0.5% NaCl, pH 8.9) did not show a degradation effect on the molecular weight of HA. Chromatography in size-differentiated gels indicated a possible molecular weight of 700,000 Da. Example 7 Drug Cytotoxicity Analysis: 弑 妫 妫 经济 经济 经济 经济 经济 经济 经济 经济 经济 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工 员工For those who want it. When 5-FU was administered therapeutically at 10.5 mg/kg (400 mg/m2) via an intravenous route (iv), the plasma level was 8/zg/ml [61/zM, within 30 min. Kubo, 1990]. Based on the plasma concentration in the living body, the concomitant drug concentrations are selected; 〇, 1, 5, 10, 20, 50, 100 // M 5_FU ΙΟΟ Μ Μ HA. The cell lines MDA-MB-468, MDA-MB-231 were cultured as MDA-MB-43 5 as described above in the specifics of Examples 4 to 5. When these fine-45- paper scales are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1284042 A7 _____B7__ V. Invention Description (43) When the cells reach the confluence, the cells are from 0 · 2 5 % The trypsin/0 · 0 5 % EDTA flask was removed. The single cell suspension was counted in a Coulter counter (ZMI model) and the cells were resuspended to 100,00 cells/ml in cell-specific medium. The cells were placed in a 24-well plate (2 cm2 surface area) by adding 〇5 ml of cell suspension per well to produce 50,000 cells/well. The culture was allowed to continue for 24 h, and the single cell layer was washed with Η B S S and the medium (5 - F U + Η A) was used before the medium was removed. After 4 days of growth in the test medium, the culture was washed with HB S S, which was removed from the well by trypsinization with 0.25% trypsin / 0.05% EDTA and counted as a Coulter Counter. Example 8 EA as a sputum delivery and sputum 麴 麴 埘 砰 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以 以The mammary epithelial cancer cell line MDA-ΜB-468 was selected as the source of this cancer cell to produce any nude mouse human tumor graft. The cells were routinely cultured and subcultured as described in Example 5. For injection into mice, the cells were cultured to 100% confluence and pelleted in 0.025% trypsin/0·01% EDTA solution, washed twice by centrifugation at 400g in a Beckman TJ-6 bench centrifuge. Counted using a Model-ZM Coulter counter and resuspended in a serum-free Leibovitz L-15 medium at χ 1〇8 cells/ml ο -46- This paper scale applies to China@家标准(CNS) Α4 Specifications (210x297 公董)" ' ---Γ-hl· Llhc----__ Imp (please read the note on the back and fill out this page) l·The Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1240442 A7 B7 Description of the Invention (44) The athymic CBA/WEHI female nude mice, 6 to 8 weeks old, are maintained under special conditions without pathogens and have arbitrarily obtained sterile food and water. Each mouse received an injection containing 5 x 106 cells in 50/zl. The cells are injected into a mammary fat pad directly below the first nipple (Lamszus et al, 199 7) with a needle of 2.6 g a g g e. Tumors were measured weekly (did2d3) by measuring three diameters at right angles to the horizontal plane. Tumor volume was estimated using the following formula: (1 / 6) 71 (1 d2d3) Treatment with 5-FU ± HA was approximately 4 to 8 weeks after cancer cell inoculation. The mean tumor size of the mice used in each study is summarized in Table 5. At the beginning of the study, the human body was swollen with cancer and the tumor volume (mean + SEM). The tumor accounted for the net body volume % (average soil SEM) 5 - FU sales 〇·41 ±〇.〇lmm3 0.22 + 0.10mm3 Validity: 6 weeks 0 · 3 7 ± 0.2 0mm3 0.19 + 0.10mm3 Validity period: 6 Η 0.61 +0.36mm3 0.28 +0.17mm3 To confirm when the breast cancer cell line MDA - Μ B - 4 6 8 was injected into a suitable animal host Tumor production and its ability to produce tumors," does not require a pathological test. Since a tumor is a pathologically viable jk tube hyperplasia (neovascularization), the microvascular network supplies nutrients to the tumor. Vascular hyperplasia, catheter invasion, necrosis, 3 —_47· ^Applicable to Chinese National Standard (CNS) A4 specification (21Qx297 public Chu) ' ' m· ^ri« ϋϋ —ϋ ϋ — ϋ (please read the notes on the back first) Fill in this page), 1Τ Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Zhang-paper 1284042 Α7 Β7 V. Invention description (45) The pattern of death, southern mitosis index and abnormal nuclear appearance are the characteristics of breast epithelial cancer . Examination of hematoxylin and eosin-stained breast cancer tumor sections revealed all of the general characteristics associated with tumor survival, thus confirming that animal hosts successfully maintained grade II human breast epithelial cancer. Two tumor-bearing mice were given a consistently dead dose of sodium pentobarbital after approximately 8 weeks of tumor induction. Within 3 minutes after killing the mice, the tumors were surgically removed and immediately fixed in 1 〇 % buffered formalin for 2 h. The fixed tumor was dehydrated in a series of 7 〇 -1 〇 〇 〇 /0 alcohol, and then embedded in paraffin, and a 2-4/zm slice was cut therefrom. The section was placed on a glass slide, dewaxed and brought into contact with water. The slides were washed in phosphate-buffered saline (PBS) for 3 x 5 minutes. The pro-isoprotein was blocked with 10% fetal bovine serum for 10 minutes and then washed with PBS. The antibody for detection was applied at room temperature (RT) for 6 minutes. The antiserum or anti-system is against RH AMM and C AE. The slides were washed in P B S for 3 X 5 minutes, and endogenous peroxidase activity was blocked by immersing them in 0.3% H 2 〇 2 /methanol for 20 minutes. After another PBS wash, peroxidase-conjugated porcine anti-free secondary antiserum (Dako, Denmark) was applied at RT for 60 minutes and then washed in PBS for 3 x 5 minutes. A sigma fast DAB (3,3 -«^ amine conjugate, Sigma, St. Louis, U.S.A) lozenge was prepared according to the manufacturer's instructions and the DAB solution was applied at room temperature for 54 minutes. The slides were washed with tap water for 10 minutes, stained with hematoxylin, dehydrated and placed. -48- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) __--ΓΓ t.----- (Please read the notes on the back and fill out this page) Employee Consumption Cooperative Printed 1240442 A7 - _B7_ V. INSTRUCTION DESCRIPTION (46) The human origin of the tumor was determined by staining the tumor and surrounding tissue with a human-specific cancer staining marker. The appearance of C E A clearly shows that it is a human tumor, but is maintained in the cardiovascular system of a mouse host. As a hypothesis via receptor-mediated internalization or binding to tumor-locked lines, it is confirmed that the expression of HA receptor, CD44 and RHAMM is essential. Table 6 lists the extent of expression of the graft recipients in human breast cancer tumors. ———ΚΙ-ΙΓ hr hl· 111 1111 11 ^ (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs Intellectual Property Office Staff Cooperatives Print -49- This paper scale applies to China National Standard (CNS) A4 Specifications (210X297 mm) 1284042 A7 B7 V. Description of invention (47) Table ^ Distribution of people who are growing in the naked 麴氪 麴氪 痼 痼 痼 经济 经济 经济 经济 经济 经济 智慧 智慧 智慧 智慧 HA HA HA HA HA HA HA HA HA HA HA HA HA HA HA HA Expression of epitopes on tumors % CD44H significantly binds and internalizes the isoform of HA (Culty et al., 1992) on cells other than some stromal cells + + + + CD 4 4 ν 6 The role in the tumor is unknown, but when used as a prognostic factor. The higher the performance, the lower the chance of survival (Friedrichs et al., 995) + CD44v3 is often overexpressed in breast cancer (Friedrichs et al., 995). RHAMM transformation and tumor detail invasion are necessary (Hall et Al., 1 995) Infiltrating tumor cell population with high expression on cells surrounding the necrotic area + + + CEA showing fetal antigens on malignant cells (Haskell, 1 990) + + + in all tumor cells + The ratio of the percentage of epitope expression on the tumor is quantified as: 0% 1 - 2 5 % 2 6 - 5 0 % 5 1 - 7 5 % 7 6 - 1 0 0 % -50-

+ + + + + + + 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 1ιϋ·.ι^ι»ι^ϋ ^Kml· —ϋ ϋϋ —ϋ ϋϋ ^1 (請先閲讀背面之注意事項再填寫本頁) 卜訂 1284042 A7 B7 五、發明説明（48) :^_11^-^1；舆11人於裸鼠模型中的使用 II— I —I— n I (請先閱讀背面之注意事項再填寫本頁) 25隻小鼠被隨機的分為二群。第1組只接受[3H]5-FU ，而第2組接受[3H]5-FU/HA結合物。該治療利用注 射注射筒在注射之前與之後的重量經由尾部血管被量化地 投藥。氣化的5-FU(平均注射dpm 士 SEM: 31，313,〇〇2±131，348)其中含有 30mg/kg 5-FU 士 12.5mg/kg HA於每一注射中被輸送。 為了確保一確定量的[3H]5-FU被輸送至血液血管中 量化’該位於尾部血管的注射處被解剖並且計量5 - f U。 被留存於注射位置的5 -FU注射之平均百分比也被決定。 被輸送至血管的5 - F U量（可用以分配至組織以及腫瘤的5 -FU)如下被計算： 被$送至於注射筒注射材料之留存於注射位 企管的5- 中量的差 Dpm/mg 置的Dpm FU 的量 一（mg) x _ (Dpm) 輸送至血管之5 - F U的數量從此之後將被稱作”注射劑量” 經濟部智慧財產局員工消費合作社印製 〇 為了正轉的比較樣品族群以及標準化於器官與各腫瘤 大小的輕微差異，於身體器官與腫瘤以及體液中的5-FU >辰度以％被注入劑量/組織克重來表示。 小鼠被個別地被置於柔軟的、非可浸濕的塑膠圍攔如 此尿液可被收集。於注射之後1 〇min、20min、3 Omin -51- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）+ + + + + + + This paper size applies to Chinese National Standard (CNS) Α4 Specifications (210Χ297 mm) 1ιϋ·.ι^ι»ι^ϋ ^Kml· —ϋ ϋϋ —ϋ ϋϋ ^1 (Please read the back first Note: Please fill out this page) Bu Biao 1284042 A7 B7 V. Invention Description (48) : ^_11^-^1; Use of 11 people in nude mouse model II—I —I— n I (Read first Precautions on the back side of this page) 25 mice were randomly divided into two groups. Group 1 received only [3H]5-FU, while Group 2 received [3H]5-FU/HA conjugate. The treatment is dosed quantitatively via the tail vessel using the injection syringe prior to and after injection. Gasified 5-FU (mean injection dpm SEM: 31, 313, 〇〇 2 ± 131, 348) containing 30 mg/kg 5-FU ± 12.5 mg/kg HA was delivered in each injection. To ensure that a certain amount of [3H]5-FU is delivered into the blood vessels, quantify the injection at the tail vessel is dissected and meters 5-f U. The average percentage of 5-FU injections that were retained at the injection site was also determined. The amount of 5-FU delivered to the blood vessels (used to distribute to the tissue and the 5-FU of the tumor) was calculated as follows: 5 to the amount of Dpm/mg left to the injection tube for the injection material injected into the syringe The amount of Dpm FU is 1 (mg) x _ (Dpm) The amount of 5 - FU delivered to the blood vessels will be referred to as the "injection dose" from the Ministry of Economic Affairs, the Intellectual Property Office, and the Consumer Cooperatives. The sample population and the slight difference in the size of the organ and each tumor were expressed in the body organs and tumors as well as in the body fluids. The 5-FU > The mice are individually placed in a soft, non-wettable plastic enclosure so that urine can be collected. 1 〇 min, 20 min, 3 Omin -51 after injection - This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm)

X 經濟部智慧財產局員工消費合作社印製 1284042 Α7 _Β7 五、發明説明（49 ) 、lh或2h，小鼠藉由腹腔注射〇·丨^1的戊巴比妥鈉被麻 醉。 當麻醉該等動物時，血液自心臟或是大血管使用針頭與注 射筒被收集。血液被收集於一 EDTA塗覆玻璃管並且於 14,000gav 離心 lOmin。在以 1〇〇"1 的 3〇%v/v 過氧化 氫脫色並且加入3ml HiSafell scintillant之後，放射 性於一 5 0 v 1等分試樣被計數。為克服化學化學發光與 光致發光，視樣品來源該樣品於一 W a 11 a c 1 4 1 0 -計 數器中重覆3、7或20 d期間被計數2 min。於被計數之 間的期間，樣品於室溫下被儲存在黑暗中。所有的計算結 果於一穩定的樣品被執行，於此所有的化能與光能螢光均 已被移除。 為了決定被注入MTX於血漿中的百分比，需要計算 出每一老鼠的總血漿體積（ml)，使用下述公式： 老鼠大小（g)x老鼠血液體積（0·07)血液的血漿部份 (0.59) 被注入的MTX於血漿中百分比接著被計算： m 1) X dpm/ml 血漿 X 100 被注入d p m總量 =%於血漿中被注入的Μ T X 。 當可能的尿液自該非可浸濕的塑膠園欄以一注射筒和 針頭被收集時。該尿液於丨4,〇〇〇gav離心1 〇 min被澄清 -52- " 本紙張又度適用中國國家檩準（CNS ) M規格（ho'〆297公羡 --.LiwL--rr-hl·---衣---I---- 訂 (請先閱讀背面之注意事項再填寫本頁} I五 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 發明説明（50) 。其放射性内含物在加入3ml HiSafell scintillant至 範圍8 - 3 0 # 1樣品中之後被量測。雖然於確切的量化每 —老鼠的尿液有一技術上的困難，5-FU劑量於尿液中的 百分比藉由下述公式被計算： 龍集時間（h) X 42以1 4 dt)m/v 1尿液X 1 00 被注入總d p m 在血液收集以頸部錯位殺死老鼠。殺死老鼠之後立刻 切下該腫瘤、肝臟、心臟、脾臟、膀胱、左右腎臟、子宮 、肺臟、胃、腸、腦以及淋巴結並且整個的分析其放射性 。於各組織中的放射性總量藉由溶解100-400mg組織於 3-6ml 的 OptiSolv (ACC，Melbourne，Australia)中 歷時3 6 h，2 2 °C來被量測。在溶液化完成後，於組織中 的放射性在加入10ml的HiSafelll scintillant之後被計 數。該樣品如先前所述的被計數以克服化學發光。 如第12圖所示者，當HA與5-FU結合時有一顯著的 鎖定效果。在1 0 m i η時於腫瘤中藥物留存的相對最大增 加被觀察到，於此該ΗΑ藉由2.42 (ρ = 〇.〇〇1 students t-test)的因子增強藥物攝取。於HA/5-FU投藥之後 2 0min及30min，於5-FU的腫瘤攝取中統計學地顯著也 被注意到，分別增加藥物攝取1 · 5倍至2倍（p < 〇 · 0 0 1，X Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 Α7 _Β7 V. Inventive Note (49), lh or 2h, mice were intoxicated by intraperitoneal injection of sodium pentobarbital in 〇·丨^1. When anesthetizing such animals, blood is collected from the heart or large blood vessels using a needle and a syringe. Blood was collected in an EDTA coated glass tube and centrifuged at 14,000 gav for 10 min. After decolorization with 1〇"1 of 3〇%v/v hydrogen peroxide and addition of 3ml HiSafell scintillant, the radioactivity was counted in a 50 volt aliquot. To overcome chemiluminescence and photoluminescence, the sample was counted for 2 min during a repeat of 3, 7 or 20 d in a W a 11 a c 1 4 1 0 -counter depending on the sample source. During the period between counts, the samples were stored in the dark at room temperature. All calculations were performed on a stable sample, and all of the chemical and light fluorescence was removed. To determine the percentage of MTX injected into the plasma, the total plasma volume (ml) of each mouse was calculated using the following formula: Mouse size (g) x mouse blood volume (0·07) Plasma fraction of blood ( 0.59) The percentage of MTX injected into the plasma is then calculated: m 1) X dpm/ml Plasma X 100 is injected into the total amount of dpm = % Μ TX injected into the plasma. When possible urine is collected from the non-wettable plastic garden column with a syringe and needle. The urine was clarified at 丨4, 〇〇〇gav centrifugation 1 〇min-52- " This paper is again applicable to China National Standard (CNS) M specification (ho'〆297羡--.LiwL--rr -hl·---衣---I---- Order (please read the note on the back and then fill out this page) I. Five Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1284042 A7 B7 Invention Description (50). The radioactive content was measured after adding 3 ml of HiSafell scintillant to a range of 8 - 30 # 1 samples. Although there is a technical difficulty in quantifying the urine of each mouse, the 5-FU dose is in the urine. The percentage is calculated by the following formula: Dragon set time (h) X 42 by 1 4 dt) m/v 1 urine X 1 00 is injected into the total dpm in the blood collection to kill the mouse with a neck dislocation. Kill the mouse Immediately thereafter, the tumor, liver, heart, spleen, bladder, left and right kidneys, uterus, lungs, stomach, intestines, brain, and lymph nodes were excised and the whole radioactivity was analyzed. The total amount of radioactivity in each tissue was dissolved by 100-400 mg. Organized in 3-6ml of OptiSolv (ACC, Melbourne, Australia) for 3 6 h, 2 2 °C After the completion of the solubilization, the radioactivity in the tissue was counted after the addition of 10 ml of HiSafelll scintillant. The sample was counted as previously described to overcome chemiluminescence. As shown in Figure 12, when HA and 5-FU There was a significant locking effect when combined. The relative maximum increase in drug retention in the tumor at 10 mi η was observed, which was enhanced by a factor of 2.42 (ρ = 〇.〇〇1 students t-test). Drug intake. Statistically significant in the 5-FU tumor uptake at 20 min and 30 min after HA/5-FU administration, respectively, increased drug intake by 1.5 to 2 times (p < 〇· 0 0 1,

Students t-test)。其他的時間點於5-FU單獨投藥及與 HA共注射之間並沒有顯示一統計學地顯著差異。 -53- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)Students t-test). The other time points did not show a statistically significant difference between 5-FU alone and co-injection with HA. -53- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back and fill out this page)

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釐 公 7 29 X -54- 1284042 A7 B7 五、發明説明（52 ) 證實諸如肝臟、脾臟以及腎臟之代謝活動器官沒有表 現一高位準的藥物鎖定是很重要的，該鎖定會妨害任何增 加增加腫瘤鎖疋的正向方面。表7例示了於不同時間點測 試[3H]5-FU在每一組織的攝取。 第1 3圖中顯示η A的初級代謝器官為肝臟、淋巴結與 脾臟，然而5 - F U大部分於肝臟中代謝。 當與HA共注射時，此等器官並沒有顯示5_?11攝取的 顯著增加。然而，在與HA投藥lOmin之後腎臟沒有顯著 的增加鎖定（增加1 · 8倍，p = 〇 · 〇 〇 4 )。於此點之後，即使 沒有統計學地顯著，增加藥物攝取持續直到樣品期間的終 點。 膀胱、腸以及骨髓並沒有顯示5_FlJ之攝取的增加。 於諸如子宮的組織中，在1〇 min時於藥物攝取有一短期 的增加（增加1.8-倍，ρ = 〇· 〇3 2)，但是於其他時間點沒有 顯示差異，所以此一觀察之顯著性尚未能確定。 於一個或二個樣品點上，胃、腦以及肺臟確實顯示弘 FU攝取的增加。然而，由於在每一個時間點的小動物數 量（η = 5)不能實質的統計顯著性’即使一可分辨的趨勢如 第1 4 a - C圖所示的可被觀察到。 於lh與2h的後者樣品點中於心臟的5_FU顯著的減少 被注意到，於此與HA共投藥造成分別59% (p==〇 以 及 53% (p = 〇.021)的減少。 由不同的排尿速度，由每一老鼠收集尿液係為不可能 的；因此足夠的尿液被收集以供統計分析。 -55- 本紙張尺度適用中國國家標準（CNS ) A4規格（2!0X297公釐） 1· ·!ϋ«ιρ— ϋϋ —^1 ϋ^— ϋ (請先閱讀背面之注意事項再填寫本頁) I訂 經 濟 部 智 慧 財 產 局 員 工 消 f 合 作 社 印 製 1284042 五、發明説明（53 ) 當5-FU與Η A被共投藥時，於循環位準中的有一較早 的減少（表7)。透明質降低的血漿5-Fu藉由55% (ρ = 0· 00 1)。如第15圖中所示，藥力動力學的血漿半生 期於接受5-FU/HA的小鼠由28min被轉換為56min。 JLfe[io :對小鼠治瘙瘙法之押竿 期間患者的狀況被重覆評估，因此 擬人類治療療法，藉由令小鼠於一 ^ 仏 TSL /，/«1 m 、 _ 一 … 因此我們為了嚐試儘可能模 於一長期的效力研究中暴露PCT 7 29 X -54-1284042 A7 B7 V. INSTRUCTIONS (52) It is important to confirm that a metabolically active organ such as the liver, spleen, and kidney does not exhibit a high level of drug locking, which may impair any increase in tumors. The positive aspect of the lock. Table 7 illustrates the uptake of [3H]5-FU in each tissue at different time points. Figure 13 shows that the primary metabolic organs of η A are the liver, lymph nodes and spleen, whereas 5 - F U is mostly metabolized in the liver. These organs did not show a significant increase in 5-11? ingestion when co-injected with HA. However, there was no significant increase in renal lock after lOmin administration with HA (an increase of 1.8 times, p = 〇 · 〇 〇 4 ). After this point, even if not statistically significant, increased drug intake continued until the end of the sample period. Bladder, intestine, and bone marrow did not show an increase in uptake of 5_FlJ. In tissues such as the uterus, there is a short-term increase in drug intake at 1 〇 min (1.8-fold increase, ρ = 〇·〇3 2), but no difference is shown at other time points, so the significance of this observation Not yet sure. At one or two sample points, the stomach, brain, and lungs did show an increase in Hong FU intake. However, since the number of small animals at each time point (η = 5) cannot be substantially statistically significant, even a resolvable trend as shown in Fig. 14 a - C can be observed. A significant decrease in 5_FU in the heart at the latter sample points of 1 h and 2 h was noted, and co-administration with HA resulted in a 59% reduction (p==〇 and 53% (p = 〇.021), respectively. The rate of urination, which is impossible to collect urine from each mouse; therefore enough urine is collected for statistical analysis. -55- This paper scale applies to the Chinese National Standard (CNS) A4 specification (2!0X297 mm) ) 1· ·!ϋ«ιρ— ϋϋ —^1 ϋ^— ϋ (Please read the notes on the back and fill out this page) I. Ministry of Economic Affairs, Intellectual Property Office, Staff, F, Co., Ltd. Printed 1284042 V. Inventions (53 When 5-FU and ΗA were co-administered, there was an earlier decrease in the circulating level (Table 7). The hyalurode-reduced plasma 5-Fu was 55% (ρ = 0·00 1). As shown in Figure 15, the pharmacokinetic plasma half-life of mice receiving 5-FU/HA was converted from 56 min to 56 min. JLfe [io: The condition of the patient during the post-mortem treatment of the mouse Being repeatedly evaluated, so the human therapeutic therapy, by making the mouse in a ^ 仏 TSL /, / «1 m, _ a ... so we are Trying to be as exposed as possible in a long-term efficacy study

示同時進行短期的與長期的治療計劃。 常用於人類乳癌的治療療法係為環磷醯胺、氨甲喋呤 以及5-氟尿嘧啶其係於28日循環的第j日以及第8日被投 藥。於人類乳癌中該治療療法的開始係為6循環，於此段 1284042 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明説明（54 盘-g丄治瘙投藥計割Show both short-term and long-term treatment plans. Therapeutic treatments commonly used in human breast cancer are cyclophosphamide, methotrexate, and 5-fluorouracil, which are administered on the jth and the 8th day of the 28th cycle. In the human breast cancer, the beginning of the treatment is 6 cycles, this paragraph 1284042 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (54 disk - g 丄 瘙 瘙 瘙 计 计 瘙

治療群 服用量 6週研究： 6個月研究： 治療療法 1 ·生理食 鹽水 0· 1ml生理食鹽 水（注射等級） 7曰循環的1 及2 28 日 及8 2.5- FU/HA 0.1ml含有 ： 3 Omg/kg 5 -FU+12.5mg/kg HA 7日循環的1 及2 及8 3. ΗΑ 0.1ml含有 ： 1 2 · 5 mg/kg H A 7曰循環的1 及2 2 8日循環的1 ;8 4. 5-FU 0 · 1 m 1含有 ： 3 Omg/kg 5-FU 7曰循環的1 及2 2 8日循環的1 及8 5 · Η Α然後 5-FU 0 · 1 m 1含有 ： 1 2.5mg/kg 或 HA30mg/kg 5-FU 7曰循環 1 ： HA 2 ： 5-FU 3 ： HA 4 : 5-FU 28 0¼¾ 1 : HA 2 · 5 - F U 8 ： HA 9 : 5-FU 6 . HA 0.1 ml含有 ： 12.5mg/kg HA 7曰循環 1 : HA 3 ： HA -----. 7.5-FU 0.1 m 1含有 ： 30mg/kg 5-FU 7曰循環 — 2 ： HA 4 ： HA 老鼠被分為7組，每組8隻動物被用於短期研究，以 及5 8隻動物用於長期研究（參考表8的劑量與投藥計劃） 〇 該療法沒有被延伸超過6個月，因為已顯示超過6個 月的治療法一般不會結合更佳的益處（Harris et al， 1992)。 ’ 如實例8所述，於長時間治療研究的治療施行日，動 物們被稱重並且計量腫瘤體積。於6週期間研究動物們每 __ ·57_ &氏張尺度適用中國國家標準（CNS ) Μ規格（21〇χ^97公釐)---------- (請先閱讀背面之注意事項再填寫本頁)Treatment group dose 6 weeks study: 6 months study: treatment therapy 1 · physiological saline 0 · 1ml physiological saline (injection grade) 7 曰 cycle 1 and 2 28 days and 8 2.5-FU/HA 0.1ml contains: 3 Omg/kg 5 -FU+12.5mg/kg HA 7-day cycle 1 and 2 and 8 3. ΗΑ 0.1ml contains: 1 2 · 5 mg/kg HA 7曰 cycle 1 and 2 2 8 cycle 1 ;8 4. 5-FU 0 · 1 m 1 contains: 3 Omg/kg 5-FU 7曰 cycle of 1 and 2 2 8 day cycle of 1 and 8 5 · Η Α then 5-FU 0 · 1 m 1 contains : 1 2.5mg/kg or HA30mg/kg 5-FU 7曰 Cycle 1: HA 2 : 5-FU 3 : HA 4 : 5-FU 28 01⁄43⁄4 1 : HA 2 · 5 - FU 8 : HA 9 : 5-FU 6. HA 0.1 ml contains: 12.5mg/kg HA 7曰 Cycle 1: HA 3 : HA -----. 7.5-FU 0.1 m 1 contains: 30mg/kg 5-FU 7曰 cycle — 2 : HA 4 : HA mice were divided into 7 groups, 8 animals in each group were used for short-term studies, and 58 animals were used for long-term studies (refer to the dose and dosing schedule in Table 8). The therapy was not extended for more than 6 months because Treatments that have been shown for more than 6 months generally do not combine better benefits (Harris et Al, 1992). As described in Example 8, the animals were weighed and the tumor volume was measured on the day of treatment for the long-term treatment study. For the 6-week study, the animals are subject to the Chinese National Standard (CNS) 每 specifications (21〇χ^97 mm) for each __·57_ & Zhang scale---------- (please read the back first) Note on this page)

1284042 A7 ______B7_ 五、發明説明（55 ) 日被稱重並且計量腫瘤體積。動物們被個別地置於—注 射盒中，並且該注射經由尾部血管被投藥。已經實驗證明 於患者對化學治療的反應中，壓力可為一主要因子 (Shackney et H1，1 978)。因此，我們確定相同數目的 小鼠被安置於每一獸籠中，每一獸籠的動物數目視試驗階 段於5_8隻中變化。 於動物因病況的進展而死亡或是當6個月（長期）或是 6週（短期）治療療法完成時，試驗終點出現。由於動物行 為的指標，該等動物隔週的由一獨立的動物行為負責人員 檢視評估疾病進展的程度。如第1 6圖中所示者，接下來 的標準被用以檢測動物已經到達必需死亡的試驗終點： 1) ·動物不吃不喝並表現引人注意的體重減輕； 2) · 腫瘤超過體重的10% (panei a); 3) · 腫瘤大小太大以致於動物無法移動（panei B) 於試驗終點該等動物們藉由注射戊巴比妥納 (60mg/ml)被麻醉，血液被收集然後以頸部錯位殺死該 等動物。 經濟部智慧財產局員工消費合作社印製 I.rmh L: ‘ 1 - I I 1 1 11 —1 (請先閲讀背面之注意事項再填寫本頁} 於6週研究的末了，腫瘤大小被決定並且如第1 7圖所 不’ 5 - F U與5 - F U / Η A療法具有比生理食鹽水組顯著地較 小的腫瘤（P = 〇 · 〇 〇 5 )。於5 - F U與H A / 5 _ F U治療群之間， 腫瘤體積;又有顯著差異’指出兩種療法於對抗初級的腫瘤 上展現相同的效力。於生理食鹽水與Η A治療群之間，初 級的腫瘤體積沒有顯著差異。在殺死老鼠之後該腫瘤、肝 _____ -58- 本紙張尺度通用中國國家標準（CNS ) A4規格（21Gx297公慶) * 一 1284042 A7 B7 五、發明説明（56 ) (請先閲讀背面之注意事項再填寫本頁) 臟、心臟、脾臟、膀胱、左腎與右腎、子宮、肺臟、胃腸 、腦以及淋巴結立刻被切下並置於4%的以〇 〇6M碟酸 pH 7.5緩衝的福馬林中以及ίο% w/v氣化鯨蠟基π比唆中 。於組織學處理之前，該組織被固定歷時16_24h。經固 定的組織1 0 0 %的酒精中被脫水並且被包埋於石蠛塊中， 由此2·4βιη切片被置於顯微鏡的玻璃载玻片上。以蘇木 精核染劑與伊紅細胞質染劑染色該組織切片以指出任何可 指出治療毒性的病理學特徵。 每一動物收集9至11個淋巴結，確保所有於腫瘤區域 的淋被收集。現今有兩種方法用以檢測淋巴結的轉移： i) 粗略器官組織的例行的蘇木精與伊紅染色 i i) 使用諸如癌胚抗原之癌症標記的免疫組織學方 法： 於本研究中轉移檢測的兩種方法同時被使用。並非所 有可獲得的C E A抗體都會與人類乳癌細胞起反應，所以 我們測試5種抗體的反應性（DAKO，Amersham and K P L ) 〇 經濟部智慧財產局員工消費合作社印製 該蘇木精與伊紅染色的淋巴結經由Dr P. Allen (經 認可的的病理學家）於此每一淋巴結以顯微鏡檢視腫瘤細 胞的出現。該C E A免疫染色的淋巴結被顯微鏡的檢視， 任何被正向地染色的被計數並且考量淋巴結的轉移。 腫瘤體積如先前實例8中所述的藉由微血管量測以及 腫瘤體積以每日或每週的基礎被檢視被計算。 -59- 平 - ί r 警 A4 a 公 7 29 1284042 A7 __ _B7_五、發明説明（57 ) 5-FU最常見的毒性效果係於腸胃道中，於此出血的 進入腸道穿孔可能發生（Martindale，1993)。每日檢視 諸如腹瀉之GI不適並且每週注意諸如體重減輕之毒性證 據。體重減輕藉由去除腫瘤重的體重被檢視，其藉由如 Shibamoto et al，1 996所引述的以lg x腫瘤體積 (cm3)來計算。為了展現該動物重量的變化，體重於治療 時間開始依下述標準化： 身...體-質量（去除腫瘤）-治療開始時身體質量（去除腫瘤） 治療開始時身體質量（去除腫瘤） xl 00 ____rr.uL.----- (請先閲讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 在沒有治療的動物群中，沒有諸如腹瀉之Gi道不適 被注意。對於每一治療療法，腸胃的毒性使用體重的減輕 (除去腫瘤重）作為指標。於每一動物死亡的時間，於體 重變化之百分比以先前所述的被計算。於經標準化的體重 在生理食鹽水、HA及5-FU治療群與5-FU/HA群治療群 之間有一統計學地顯著差異（第1 8圖）。接受5-FU/HA佐 劑治療法的小鼠顯示1 6%的體重增加（stu(ients t-test， p - 0 · 0 2 5 )在全部期間該未治理群組於重量表現一 2 %的淨 增加。 癌胚抗原（C E A )之免疫組織學與傳統的診斷病理提 供了一優異的淋巴結轉移量化。老鼠平均含有15-1 9個淋 巴結（Lamszus et al，1 997)，因此此一研究檢視該等 動物60-70%的淋巴結。細心的步驟接下來為了確保所有 的淋巴結排出該胞腺脂肪墊以及頰部被移除並被檢視。如 表9A顯示，所有的動物顯示淋巴結轉移。1284042 A7 ______B7_ V. INSTRUCTIONS (55) The day was weighed and the tumor volume was measured. The animals were individually placed in a injection box and the injection was administered via the tail vessels. Pressure has been shown to be a major factor in the response of patients to chemotherapy (Shackney et H1, 1978). Therefore, we determined that the same number of mice were placed in each cage, and the number of animals in each cage varied from 5 to 8 depending on the test period. The end of the test occurs when the animal dies due to the progression of the condition or when the 6-month (long-term) or 6-week (short-term) treatment is completed. Due to animal performance indicators, these animals were examined by an independent animal behavioral responsible person to assess the extent of disease progression. As shown in Figure 16, the next criteria are used to test the end of the test where the animal has reached the required death: 1) • The animal does not eat or drink and exhibits an attractive weight loss; 2) • The tumor exceeds the weight 10% (panei a); 3) · The tumor size is too large for the animal to move (panei B) at the end of the test. The animals were anesthetized by injection of pentobarbital (60 mg/ml) and the blood was collected. The animals are then killed by a dislocation of the neck. I.rmh L: ' 1 - II 1 1 11 -1 (Please read the notes on the back and fill out this page first) At the end of the 6-week study, the tumor size was determined and Figure 1 7 shows that '5-FU and 5-FU/Η A therapy have significantly smaller tumors than the saline group (P = 〇·〇〇5). 5 - FU and HA / 5 _ FU There was a significant difference between the treatment groups, tumor volume; and the two treatments showed the same efficacy against the primary tumor. There was no significant difference in primary tumor volume between the physiological saline and the ΗA treatment group. After the dead mouse, the tumor, liver _____ -58- The paper size is the common Chinese national standard (CNS) A4 specification (21Gx297 public celebration) * One 1284042 A7 B7 V. Invention description (56) (Please read the note on the back first) Fill in this page) Dirty, heart, spleen, bladder, left and right kidney, uterus, lung, gastrointestinal, brain and lymph nodes were immediately cut and placed in 4% of formalin buffered with 6M dish acid pH 7.5 and Ίο% w/v gasification cetyl group π than 唆. Prior to the treatment, the tissue was fixed for 16-24 hours. 100% of the fixed tissue was dehydrated and embedded in the stone block, whereby the 2·4βιη slice was placed on a glass slide of the microscope. The tissue sections were stained with hematoxylin nuclear stain and eosin stain to indicate any pathological features that could indicate therapeutic toxicity. Each animal collected 9 to 11 lymph nodes, ensuring that all lymph nodes in the tumor area were collected. There are two methods for detecting lymph node metastasis: i) routine hematoxylin and eosin staining of coarse organ tissues ii) immunohistological methods using cancer markers such as carcinoembryonic antigens: metastatic detection in this study Both methods are used at the same time. Not all available CEA antibodies react with human breast cancer cells, so we tested the reactivity of five antibodies (DAKO, Amersham and KPL). The Ministry of Economic Affairs, the Intellectual Property Office, the Consumer Cooperative, printed the hematoxylin and eosin stains. The lymph nodes were examined microscopically for the appearance of tumor cells via Dr. P. Allen (approved pathologist). The C E A immunostained lymph nodes were examined by microscopy, and any positively stained counts were counted and the lymph node metastasis was considered. Tumor volume was calculated as described in Example 8 above by microvascular measurements and tumor volume was examined on a daily or weekly basis. -59- 平- ί r Police A4 a Male 7 29 1284042 A7 __ _B7_ V. Description of invention (57) The most common toxic effect of 5-FU is in the gastrointestinal tract, where bleeding into the intestinal perforation may occur (Martindale , 1993). Daily review GI discomfort such as diarrhea and weekly attention to toxicity evidence such as weight loss. Weight loss was examined by removing the weight of the tumor, which was calculated as lg x tumor volume (cm3) as quoted by Shibamoto et al, 1996. In order to show the change in weight of the animal, the body weight is standardized as follows at the time of treatment: body...body-quality (removing the tumor)-body mass at the beginning of treatment (removing the tumor) body mass at the beginning of treatment (removing the tumor) xl 00 ____rr.uL.----- (Please read the note on the back and fill out this page.) The Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative is printed in the untreated fauna, and no Gi road discomfort such as diarrhea is noticed. For each treatment therapy, gastrointestinal toxicity was used as an indicator of weight loss (excluding tumor weight). At the time of death of each animal, the percentage change in body weight was calculated as previously described. Normalized body weight There was a statistically significant difference between the saline, HA, and 5-FU treatment groups and the 5-FU/HA group treatment group (Fig. 18). Mice receiving 5-FU/HA adjuvant therapy showed a 6% gain in weight (stu(ients t-test, p - 0 · 0 2 5 ). The untreated group showed a 2% weight gain over the entire period. The net increase in carcinoembryonic antigen (CEA) immunohistochemistry and traditional diagnostic pathology provides an excellent quantification of lymph node metastasis. The mouse contains an average of 15-1 9 lymph nodes (Lamszus et al, 1 997), so this study 60-70% of the lymph nodes were examined for these animals. Careful steps were followed to ensure that all lymph nodes were drained from the cell fat pad and the cheeks were removed and examined. As shown in Table 9A, all animals showed lymph node metastasis.

、tr 1284042 A7 B7 五、發明説明（58 ) 第1 9 A圖顯示淋巴結關聯的百分比（每一動物淋巴結 轉移的速度）大大地受到5_FU、5-FU/I-IA以及HA治療 的影響，於此該生理食鹽水群顯示淋巴結關聯量的顯示一 6倍的增加。再一次的，Ha顯示其可具有治療上的價值 〇 雖然於本研究的終點解剖該動物每一組織被微觀地並 巨觀地檢視腫瘤結。使用接受HA/5-FU或HA療法的小鼠 的預期’新腫瘤在鄰近於初級的腫瘤的頸部或是下臂部區 域被觀察到。併入HA至該治療療法中避免了新腫瘤形成 ，如第19B圖所示者。 於未接受治療之全部患者存活率沒有顯示一顯著差異 (表9 A)，於此來自所有群的動物完成了該治療療法。 伴隨5 - F U治療的主要毒性係為骨髓的降低以及於白 血球細胞急降評估任何治療伴隨的血液毒性係為必要的。 ^麻醉該荨動物時’血液自心臟或是大血管使用針頭以及 注射筒被收集。白血球細胞數量藉由製造一血液於老鼠生 理食鹽水（M)中之1/1〇的稀釋液，並且將之於一血球計 數器中計數。一不同的血液計數藉由計數嗜中性白血球、 淋巴球以及紅血球被計數。該等血液細胞次族群的估量與 經刊行的老鼠血液資料相比較。 -61- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 --stvTr 1284042 A7 B7 V. INSTRUCTIONS (58) Figure 19 A shows that the percentage of lymph node association (the rate of lymph node metastasis in each animal) is greatly affected by 5_FU, 5-FU/I-IA, and HA treatment. This physiological saline group showed a 6-fold increase in the amount of lymph node association. Again, Ha shows that it can be therapeutically valuable 〇 although each tissue of the animal was dissected at the end of the study and the tumor was microscopically and macroscopically examined. The expected 'new tumors' of mice receiving HA/5-FU or HA therapy were observed in the neck or lower arm region adjacent to the primary tumor. New tumor formation is avoided by incorporating HA into the therapeutic therapy, as shown in Figure 19B. Survival rates for all patients who did not receive treatment did not show a significant difference (Table 9 A), where animals from all groups completed the treatment. The primary toxicity associated with 5-F U treatment is the reduction of bone marrow and the assessment of hematopoietic toxicity associated with any treatment with leukocyte blast. ^ When the animal is anesthetized, blood is collected from the heart or large blood vessels using a needle and a syringe. The number of white blood cell cells was counted by making a dilution of 1/1 〇 of blood in the saline solution (M) of the mouse and counting it in a blood cell counter. A different blood count is counted by counting neutrophils, lymphocytes, and red blood cells. The estimates of these blood cell subpopulations are compared to the published blood data of the mice. -61- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (please read the note on the back and fill out this page) Printed by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs --stv

1284042 A7 B7 五、發明説明（ \7 9 5 經濟部智慧財產局員工消費合作社印製1284042 A7 B7 V. INSTRUCTIONS ( \7 9 5 Printed by the Consumers' Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs

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^cs;aAPH-「ZJ -..L^L--r-l·—^.---__ (請先閲讀背面之注意事項再填寫本頁) I 丁 . 、言 丨S- 1284042 A7 B7 五、發明説明（6〇 ) 為了確保並沒有誘生器官萎縮或是擴大，該等器官於 後死亡期間被移出並稱重。每一器官的質量以一全部淨重 的百分比被計算，並且與僅使用生理食鹽水群組（第丨群) 的器官相比較。 實例11長期的治瘵：6_個月痿法 當此一實驗被持續進行，有顯著的資料被產生。 TDT係為一腫瘤於大小或是細胞數目倍增所耗費的 時間，一腫瘤生長的參數其係簡單的被計量並且於觀念上 可輕易的與臨床的腫瘤狀態相關（Shackney et al5 1 9 7 8 )。藉由檢視腫瘤的倍增時間，評估腫瘤化學療法 的反應係為可能者’因為緩慢的腫瘤生長即表示對化學、、么 療法的極差反應（Schabel，1 975)。 於每一治療中該腫瘤倍增時間示於表93中。 __,ίΓΊ--f-n.----- (請先閲讀背面之注意事項再壤、寫本頁j I訂 經濟部智慧財產局員工消費合作社印製 -63-^cs;aAPH-"ZJ-..L^L--rl·-^.---__ (Please read the notes on the back and fill out this page) I D., 丨S- 1284042 A7 B7 V. DESCRIPTION OF THE INVENTION (6〇) In order to ensure that no induced organ atrophy or enlargement, the organs are removed and weighed during post-mortem. The mass of each organ is calculated as a percentage of the total net weight, and only physiologically used Comparison of the organs of the saline group (Dijon group). Example 11 Long-term treatment: 6_month 痿 method When this experiment was continued, significant data were generated. TDT is a tumor in size or It is the time it takes to multiply the number of cells. A parameter of tumor growth is simply measured and conceptually related to clinical tumor status (Shackney et al 5 1 9 7 8). By examining the doubling time of the tumor Assessing the response of tumor chemotherapy is possible because 'slow tumor growth indicates a poor response to chemistry, and therapy (Schabel, 1 975). The tumor doubling time is shown in Table 93 for each treatment. __,ίΓΊ--fn.----- (please read the back first) Note: Re-soil, write this page j I book Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing -63-

1284042 A7 B7 五、發明説明（61 ) 經濟部智慧財產局員工消費合作社印製 19 鉍车叫辱9 :蝌柘«飨尊^|崦划》+ 1|雄却赛坧飨女斓每級v农丧谁«雄vffl/ndt-s : a6<1284042 A7 B7 V. Description of invention (61) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 19 叫 叫 9 9 9 9 9 9 9 9 飨 飨 飨 蝌柘 蝌柘 蝌柘 蝌柘 蝌柘 + + + + + + + + + + + + + + + + + + + + + + + Who is the farmer? «雄vffl/ndt-s : a6<

腫瘤病理學：觀察 沒有特別特徵 沒有特別特徵 濰=飨 ^ ^ 〇4 *33嘀均 1*0傾祝 ^ < W 小面積的壞死與痂形成 沒有特別特徵 試驗終點 碟 ^ in 卜 （N 00 T-H 丧 燕 in (N 卜 Ό cn 飨 m 步表 in (Ν ϊ> 1 0 0 %無法移動 41 ^ in ^Τί t> <N co TDT 平均土 SEM 13.0±4.0 22·7±2·9 28 ±2.6 26 ± 1.75 m v〇 o +1 m 治療 生理食鹽水 5-FU 5-FU/HA HA HA接著5-FU (請先閱讀背面之注意事項再填寫本頁)Tumor pathology: no special features were observed without special features 潍=飨^ ^ 〇4 *33嘀1*0 倾祝^ < W Small area of necrosis and sputum formation No special characteristics test end point ^ in Bu (N 00 TH 燕燕in (N Ό Ό 飨 step in (Ν ϊ> 100% cannot move 41 ^ in ^Τί t><N co TDT average soil SEM 13.0±4.0 22·7±2·9 28 ±2.6 26 ± 1.75 mv〇o +1 m Treatment of normal saline 5-FU 5-FU/HA HA HA followed by 5-FU (please read the notes on the back and fill out this page)

本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 一 64 一 1284042 A7 B7 五、發明説明（62) 於5-FU/HA以及5-FU治療之間的TDT並沒有一顯 著的差異。因為於該6週期間研究，HA的投藥同時也顯 不初級的腫瘤中治療上的效果，相對於生理食鹽水之 13士4日的TDT，其展現一 26±175的TDT。在51?11之 則2 4 h投藥Η A顯然妨礙了 5 - F U腫瘤生長遲滯時間的關聯 〇 於檢視腫瘤腫瘤治療反應與腫瘤進程時，質量與體積 係為一有用的參數，但是無法有效的展示由一療法引起的 細胞毒性的效果。我們為證實HA/5FU療法是否殺死較多 腫瘤細胞以及該細胞的地點。死亡的細胞可以被病理學地 彰明： 0· 核不完整（萎縮） i i) · 細胞溶解（壞死） 將整個腫瘤圖像掃描至一 MCID電腦中，其計算整 個腫瘤量化死亡細胞的數目。具有碎片細胞核的細胞或是 溶解的細胞被框出輪廓並且掃描，此等確實區域接下來被 計算死亡的細胞。該腫瘤百分比被歸因於死亡細胞如下被 計算： 經濟部智慧財產局員工消費合作社印製 蒌縮以及壞死細胞區域X 1 〇 〇 整個乳癌腫瘤區域 一可生存的細胞較衰亡中或是死去的細胞含有較多的 水份’因此藉由測定該乾腫瘤質量對濕潤腫瘤質量的比例 ’預測可生存的對非可生存的細胞的區域係為可能的。 該腫瘤被對稱的切開，於此一半用於腫瘤病理學的處理， -65 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公襲)' ' 1284042 A7 五、發明説明（63 ) 而另一半測量其在50°C中乾燥48h前後的重量。該乾燥腫 瘤質量的百分比如下被計算： (請先閲讀背面之注意事項再填寫本頁) 乾燥腫瘤質詈y irm 濕潤乳癌腫瘤質量 所有患者的存活時間以該動物於開始治療後存活的時 間（日或是週）來被計算。 每一治療的腫瘤倍增時間於表9八顯示。在5_fu/ha 以及5_FU治療之間的TDT沒有顯著差異。然而，HA的 投藥於初級的腫瘤上顯示一治療上的效果，於此該tdt 顯著的大於生理食鹽水群（p，〇 〇5， MultipleThis paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm). One 64 One 1284042 A7 B7 V. Inventive Note (62) The TDT between 5-FU/HA and 5-FU treatment is not significant. difference. Because of the 6-week study, the administration of HA also showed a therapeutic effect in the primary tumor, which showed a TDT of 26 ± 175 compared to the TDT of the normal saline. In the case of 51?11, 24 hours of administration, ΗA obviously hindered the correlation of 5 - FU tumor growth lag time. When examining tumor tumor treatment response and tumor progression, mass and volume were a useful parameter, but could not be effective. Shows the effect of cytotoxicity caused by a therapy. We confirmed whether HA/5FU therapy kills more tumor cells and the location of the cells. Dead cells can be pathologically revealed: 0. Nuclear incomplete (atrophy) i i) · Cell lysis (necrosis) The entire tumor image is scanned into a MCID computer, which counts the number of dead cells quantified throughout the tumor. Cells with fragmented nuclei or lysed cells are framed and scanned, and these positive regions are then counted as dead cells. The percentage of tumors attributed to dead cells is calculated as follows: Department of Economic Intelligence, Intellectual Property Bureau, Staff Consumption Cooperative, Printed Contracture, and Necrotic Cell Area X 1 〇〇 Whole Breast Cancer Tumor Area A viable cell is more dead or dead. It is possible to predict the survival of a region of non-viable cells by measuring the ratio of the dry tumor mass to the mass of the wet tumor by including more water. The tumor is symmetrically cut, and this half is used for the treatment of tumor pathology. -65 - This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 public attack) ' ' 1284042 A7 V. Inventive Note (63) The other half was measured for its weight before and after drying at 50 ° C for 48 h. The percentage of dry tumor mass is calculated as follows: (Please read the note on the back and fill out this page) Dry tumor quality 詈 irm Wet breast cancer tumor quality The survival time of all patients is the time the animal survived after starting treatment (day Or week) to be calculated. The tumor doubling time for each treatment is shown in Table 9-8. There was no significant difference in TDT between 5_fu/ha and 5_FU treatment. However, administration of HA on a primary tumor shows a therapeutic effect, where the tdt is significantly greater than the physiological saline population (p, 〇 〇 5, Multiple

comparison Tukey test)。在 5-FU 之前 24h 投藥 HA 顯示一治療上的妨礙任何5-FU腫瘤生長的遲滯時間。 引用的用於5-FU之治療時間為26% (lnaba et al， 1 9 8 9 ) ’但疋該等接受5 - F U的動物並沒有表現“治癒” 。兩隻接受H A / 5 - F U佐劑治療法的小鼠表現“治癒”， 於此該腫瘤在治療大約i 2週之後完全的壞死並掉落。第 經濟部智慧財產局員工消費合作社印製 2 0B圖顯示於接受以HA以及HA/5_FU治療的小鼠顯示 有小痂形成的特徵，而第20C圖顯示在大面積的痂形成之 後有小規模的壞死。小鼠之一經歷了小節的重新生長，但 是第二隻老鼠於22週時仍然沒有腫瘤。如於第2〇a圖中 所見，接受5-FU或是生理食鹽水的小鼠具有大的腫瘤， 由於腫瘤質量的緣故其最基本的結束該動物的死亡（表 9B)，但是接受HA + 5-FU的小鼠顯示一腫瘤外觀特徵， 於此一小面積的壞死於一痂之後形成。 -66- 本紙張尺度適用中國國家標準（CNs ) a4規格（210X297公釐） 1284042 A7 B7 五、發明説明（64 ) 於第22週，5-FU/HA小鼠的37·5%依然存活，並 且對於該等5 - F U / Η Α佐劑治療法小鼠，主要的死因是體 重減輕以及代謝壓力（表9 B )。 當HA 土 5-FU被投藥至帶有人類乳癌外移植物的小 鼠時’於所有的患者生存率中呈現一顯著差異。於24週 研究的第22週，唯一存活的群是5_fu/HA以及HA，如 第2 1圖中所示者。 結論 得自5 - F U鎖定的數據顯示當5 f u與Η A於時間點1 〇 、20、3 0分鐘共注射時，分別相對於5-FU攝取2.4、 1 · 5及2倍的統計學的顯著增加的增加。這指出5 -FU藉由 ΗA被結合至腫瘤上。有兩個可能的ha鎖定5_Fu至腫瘤 的機制係為可能者： HA包含結合5-FU結合至該等受體（CD44)並且藉 由受體-調節胞飲被内化，如此釋放藥物至該腫瘤細胞中 ll.fi—φΐ (請先閱讀背面之注意事項再填寫本頁) I1Τ 經濟部智慧財產局員工消費合作社印製 ΗA分子篩網將會成為一向外擴散的障礙，如此於 HA結合至受體（CD 44及RHAMM)之後，該被夾帶 5 - F U可以擴散至腫瘤細胞中。然而藉由η a基質被留 於該細胞表面，5 _ F U對於一般被利用以5 _ F U運送至細 中主動運送具有增加可獲取性。 HA的代謝主要於淋巴結（Fraser et al，1 9 8 8)以 肝臟（Laurent et al，1986)中發生。HA —般由血管 藉由受體·調節的細胞的攝取以及於肝臟（8 〇 _ 9 〇 % )、 的 存 胞 及 中 腎 -67· 1284042 A7Comparison Tukey test). Administration of HA at 24 h prior to 5-FU showed a therapeutic lag time that prevented any 5-FU tumor growth. The cited treatment time for 5-FU was 26% (lnaba et al, 1 9 8 9 '' but the animals that received 5 - F U did not show "cure". Two mice receiving the H A / 5 - F U adjuvant treatment showed "cure", where the tumor completely necrotic and fell after approximately i 2 weeks of treatment. The Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative printed 20B shows that the mice receiving HA and HA/5_FU showed the characteristics of small sputum formation, while the 20C chart showed that there was a small scale after the formation of large area sputum. Necrosis. One of the mice experienced a re-growth of the bar, but the second mouse still had no tumor at 22 weeks. As seen in Figure 2, mice receiving 5-FU or saline had large tumors that ended the animal's death due to tumor quality (Table 9B), but received HA + 5-FU mice showed a tumor appearance characteristic, and a small area of necrosis was formed after one sputum. -66- This paper scale applies to Chinese National Standard (CNs) a4 specification (210X297 mm) 1284042 A7 B7 V. Inventive Note (64) At week 22, 37.5% of 5-FU/HA mice are still alive, And for these 5 - FU / Η Α adjuvant treatment mice, the main causes of death were weight loss and metabolic stress (Table 9 B ). When HA soil 5-FU was administered to mice with human breast cancer explants, there was a significant difference in survival rates among all patients. At week 22 of the 24 week study, the only surviving group was 5_fu/HA and HA, as shown in Figure 21. Conclusion The data obtained from 5-FU lock showed that when 5 fu and Η A were co-injected at time points of 1 〇, 20, and 30 minutes, they were ingested 2.4, 1.5, and 2 times, respectively, relative to 5-FU. Significantly increased increase. This indicates that 5-FU is bound to the tumor by ΗA. There are two possible mechanisms for ha-locking 5_Fu to tumors: HA contains binding to 5-FU to these receptors (CD44) and is internalized by receptor-regulated pinocytosis, thus releasing the drug to the Ll.fi-φΐ in tumor cells (please read the notes on the back and fill out this page) I1Τ The Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs printed the ΗA molecular sieve will become an obstacle to the outward spread. After the body (CD 44 and RHAMM), the entrained 5-FU can diffuse into the tumor cells. However, by the η a matrix being left on the cell surface, 5 _ F U has increased accessibility for the active transport that is generally utilized to transport 5 _ F U to fine. The metabolism of HA occurs mainly in the lymph nodes (Fraser et al, 798) in the liver (Laurent et al, 1986). HA is generally taken up by blood vessels by receptor-regulated cells and in the liver (8 〇 _ 9 〇 % ), and in the kidneys -67· 1284042 A7

五、發明說明（65 ) (請先閱讀背面之注意事項再填寫本頁) 臟（10%)、脾臟（0.1%)和骨髓（〇.1%)中被澄清（Fraser et al，1 983)。循環HA藉由全部的代謝受體，同時也被 肝臟内皮細胞（LEC)受體（Eriksson et al，1 9 83 )被 攝取，於此該CD44受體呈現與HA内化結合至細胞的 步驟有涉，而非新陳代謝，然而RHAMM受體僅與細胞 運動。結合HA與5-FU可造成高位準的5邛1；被鎖定至 該Η A或5 - F U新陳代謝的位置。得自鎖定實驗的數據（表 6)顯示當與HA投藥時，於5 _FU鎖定至該肝臟有一顯著 的增加。因為沒有增加的鎖定至該肝臟被發現，其暗示該 位於肝臟的LEC受體相對於CD44受體具有一較低的親和 力。另一個可能性係為CD44異構體於肝臟的表現 (Stamenkovic et al，1991)並不會以一高親合性結和 至HA。因為藉由肝臟沒有增加的5-FU鎖定於其他如脾 臟、骨髓以及淋巴結之代謝器官中被注意到。 經濟部智慧財產局員工消費合作社印製 在10分鐘的時間内，於5-FU鎖定至腎臟有一顯著的 1.8倍的增加。雖然其他的4個時間點，當ha/5-FU被 共投藥於5-FU攝取藉由腎臟沒有顯示一顯著的增加，於 此顯示 般的趨勢發生，於以與HA投藥此多於5-FU 輸送至腎臟。用以最終消除％FU的主要路徑係由體内以 泌尿器官排出。即使於藥物與Η A的腎臟内含物中似乎有 一點增加，但是我們相信由其它的證據，HA藉由腎臟被 攝取並且快速的被代謝，如此其留存時間將會很短暫並結 合至藥物將會很快旳被釋放至尿液中。 -68- 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐） 1284042 A7 B7 五、發明説明（66 ) 於諸如胃、腦、肺臟以及子宮之組織，短期的鎖定於 -個或多個時間點被注意到。沒有_致的藥學動力學模式 被產生，其將可指出我們需要增加新的族群樣品數目，其 可清楚的指出此等觀察結果是否真冑。於在1〇 3〇_内 增加至腦之敎的案例中’其將可藉由先前的觀察被解釋 。ha已經被結合用以增強藥物通過血液腦障壁之能力 (Nelson & Falk, 1 994)。 於30 min以及i h的時間點，5邛1;與11八共投藥於 肺臟中有一顯著的增加。已經報導，肺臟巨噬細胞含有高 位準的 HA-結合、CD44 異構體（Underhill et al 1993) ，其可達成增加的鎖定。此可被結合至一治療上的優點於 肺臟上皮癌的治療結合，於此小細胞及大細胞肺臟上皮癌 已經報導含有CD44與RHAMM的過度表現（Horst et al，1 990)。 在1及2h時，當HA與5-FU共注射時，鎖定至心臟 的5-FU顯著減少。因為5_FU投藥可能會產生一心臟毒V. INSTRUCTIONS (65) (Please read the note on the back and fill out this page) The viscera (10%), spleen (0.1%) and bone marrow (〇.1%) are clarified (Fraser et al, 1 983) . Circulating HA is taken up by all metabolic receptors and also by the liver endothelial cell (LEC) receptor (Eriksson et al, 199 83), where the CD44 receptor exhibits binding to HA internalization to cells. It involves, but not metabolism, whereas the RHAMM receptor only moves with cells. Binding to HA and 5-FU can result in a high level of 5邛1; it is locked to the position of the ΗA or 5-F U metabolism. Data from the lock-in experiment (Table 6) showed a significant increase in 5 _FU lock to the liver when administered with HA. Since no increased lock was found in the liver, it suggests that the LEC receptor located in the liver has a lower affinity relative to the CD44 receptor. Another possibility is that the expression of the CD44 isoform in the liver (Stamenkovic et al, 1991) does not bind to HA with a high affinity. This is noted because the 5-FU that is not increased by the liver is locked in other metabolic organs such as the spleen, bone marrow, and lymph nodes. Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative Printed In a 10-minute period, there was a significant 1.8-fold increase in the 5-FU lock to the kidneys. Although the other 4 time points, when ha/5-FU was co-administered in 5-FU uptake by the kidney did not show a significant increase, this shows a tendency to occur, in order to do this with HA more than 5- FU is delivered to the kidneys. The main pathway used to eventually eliminate %FU is excreted in the body by the urinary organs. Even though there seems to be a slight increase in the kidney contents of the drug and ΗA, we believe that by other evidence, HA is taken up by the kidneys and metabolized quickly, so that the retention time will be short and combined with the drug. Will soon be released into the urine. -68- This paper size is applicable to China National Standard (CNS) A4 specification (21〇x 297 mm) 1284042 A7 B7 V. Invention description (66) For short-term lock-in in tissues such as stomach, brain, lung and uterus One or more points in time are noted. No pharmacokinetic model is produced, which will indicate that we need to increase the number of new ethnic samples, which clearly indicates whether these observations are true. In the case of increasing to 1% within 3〇_, it will be explained by previous observations. Ha has been combined to enhance the ability of drugs to pass through the blood barrier (Nelson & Falk, 994). At the time of 30 min and i h, there was a significant increase in the lungs with 5邛1; It has been reported that lung macrophages contain high levels of HA-binding, CD44 isoforms (Underhill et al 1993), which achieve increased locking. This can be combined with a therapeutic advantage in the therapeutic binding of lung epithelial cancer, which has been reported to contain excessive expression of CD44 and RHAMM (Horst et al, 1 990). At 1 and 2 h, 5-FU locked to the heart was significantly reduced when HA was co-injected with 5-FU. Because 5_FU administration may produce a heart poison

性（MIMS，1 997)，相較於5-Fu單獨投藥，5-FU與HA 經濟部智慧財產局員工消費合作社印製 的投藥可能會降低對心臟毒性的程度。 當評估HA/5-FU佐劑治療法的療效時，數個觀察在 長期和短期計劃中被一致化。 接受HA/5-FU或單獨HA的小鼠顯示較有活力並維 持或是增加體重，於6個月研究中，HA/5-FU小氡增加 的存活時間支持該等觀察。 -69- 本紙張尺度適用中國國家標準（CNS ) M規格（21〇><297公釐） 1284042 A7 _____B7 __ 五、發明説明（67 ) 接受5-FU/HA或HA之小鼠的腫瘤發展最大的壞死區 域，至其極限有兩個腫瘤掉落。 Η A對5 - F U的加入並未呈現一顯著的效果於初級治療 的體積，當該治療法被投藥歷時6個星期，但這不是肇因 於該腫瘤的vasculature。腫瘤含有三個區域。當HA與/ 不與5-FU共投藥時，其會到達該腫瘤處，進入該血管化 並且半壞死的區域，該被危害的血管裂縫接合。由於HA 對吸收水份的能力，其可產生一細胞外液體湧入至該腫瘤 的壞死區域，接下來增加腫瘤體積並進一步對腫瘤血管造 成危害。此一假說與該使用HA 士 5-FU確實會展現一壞死 與腫瘤細胞間液漏泄的觀察結果一致。當計算壞死細胞對 可生存的細胞區域與該乾燥：濕潤質量比被完成時，我們 可以證實這項假說。 該長期的效果研究顯示如第21圖所示者，接受 H A / 5 - F U治療的小鼠顯示一增加的存活率。此等小鼠之 平均腫瘤體積相較於其他治療群也顯示被降低。於長效期 研究中造成HA/5-FU群死亡的原因主要是由於代謝壓力 ，而造成5-FU群的死亡的原因多半是腫瘤尺寸太大造成 無法移動。所有僅使用HA的小鼠死於腫瘤太大造成無法 行動，而非由於毒性。因此HA劑量並未顯示任何毒性效 果被投藥。由該鎖定的結果其可發現5_Flj增加的鎖定發 生於當HA與5-FU結合至該腫瘤時（第12圖）。經由Ha 結合至HA特定受體CD44以及RHAMM，HA鎖定5_FU 至腫瘤的能力，其已經展現一於腫瘤位置增加的量 n.iLI-lr fihr I — — —丨 (請先閲讀背面之注意事項再填寫本頁) I訂 經濟部智慧財產局員工消費合作社印製 -70-Sex (MIMS, 1 997), compared to 5-Fu alone, 5-FU and HA Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperatives may reduce the degree of toxicity to the heart. Several observations were consistent in long-term and short-term plans when assessing the efficacy of the HA/5-FU adjuvant therapy. Mice receiving HA/5-FU or HA alone showed viability and maintenance or increased body weight. In the 6-month study, the increased survival of HA/5-FU sputum supported these observations. -69- This paper scale applies to Chinese National Standard (CNS) M specification (21〇><297 mm) 1284042 A7 _____B7 __ V. Description of invention (67) Tumors of mice receiving 5-FU/HA or HA The largest necrotic area was developed, with two tumors falling to its limit. The addition of ΗA to 5-F U did not present a significant effect on the volume of primary treatment, when the treatment was administered for 6 weeks, but this was not due to the vasculature of the tumor. The tumor contains three regions. When HA is administered with/without 5-FU, it will reach the tumor and enter the vascularized and semi-necrotic area where the compromised blood vessel cracks. Due to the ability of HA to absorb water, it can produce an extracellular fluid that floods into the necrotic area of the tumor, which in turn increases the tumor volume and further jeopardizes tumor blood vessels. This hypothesis is consistent with the observation that the use of HA 5-FU does show a necrosis and leakage of tumor cells. We can confirm this hypothesis when calculating the necrotic cell versus viable cell area and the dry: wet mass ratio is completed. This long-term effect study showed that mice treated with H A / 5 - F U showed an increased survival rate as shown in Fig. 21. The mean tumor volume of these mice was also shown to be reduced compared to other treatment groups. The cause of death of the HA/5-FU group in the long-term study was mainly due to metabolic stress, and the cause of death of the 5-FU group was mostly due to the large size of the tumor and the inability to move. All mice that use only HA die from tumors that are too large to act, not because of toxicity. Therefore, the HA dose did not show any toxic effects being administered. As a result of this locking, it was found that the increased locking of 5_Flj occurred when HA and 5-FU were bound to the tumor (Fig. 12). HA binds to HA-specific receptor CD44 and RHAMM, HA locks the ability of 5_FU to tumor, which has shown an increase in tumor location n.iLI-lr fihr I — —丨 (please read the notes on the back first) Fill in this page) I. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -70-

經濟部智慧財產局員工消費合作社印製 1284042 五、發明説明（68 ) (Culty et al，· 1 994; Wang et al Η%)，可以反應 HA與5 FU以及其他細胞毒性的藥物的使用可能有助於 克服/又有足夠藥物到達腫瘤以具有任何治療上的影響的問 題。於該短期研究中同時也發現該5-FU/HA小鼠相較於 所有其他群組顯示一體重上的增加（第2 1圖）。因此， ΗA對腫瘤位置的鎖定能力可能會減少5-FU到諸如腸之 其他器官的能力，並且降低伴隨5 _Flj治療法特別是腸胃 毒性的副作用。 相較於早先於實例1至3中所揭露的MTX/HA佐劑治 療法之預測，我們注意到一些明顯共同的結果及差異，其 摘述於表1 0中。 _____ -71- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁)Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1284042 V. Invention description (68) (Culty et al, · 1 994; Wang et al Η%), can react with HA and 5 FU and other cytotoxic drugs may have Helps overcome/have enough drugs to reach the tumor to have any therapeutic effects. It was also found in this short-term study that the 5-FU/HA mice showed an increase in body weight compared to all other groups (Fig. 21). Therefore, the ability of ΗA to lock the tumor site may reduce the ability of 5-FU to other organs such as the intestine, and reduce the side effects associated with 5 _Flj treatment, particularly gastrointestinal toxicity. In contrast to the predictions of MTX/HA adjuvant therapy as disclosed earlier in Examples 1 to 3, we noted some clearly common results and differences, which are summarized in Table 10. _____ -71- This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back and fill out this page)

1284042 A7 _______B7_ 五、發明説明（69 ) 表！！_:使用MTj^A及5-FU/HA佐«治瘙法之Μ的共同 发果以及差異 研究 類似結果 不同結果 ΜΤΧ/ΗΑ : 6個月 對 5-FU/HA ·· 6 週 接受HA /藥物的小 鼠顯示明顯的體重 增加；於患者存活 並無顯著的增加； 抑制新腫瘤形成； 展現一較長的TDT ;降低淋巴結轉移 ΜΤΧ/ΗΑ : 6個月 對 5-FU/HA : 6個月 展現一較長的TDT 5-FU小鼠展現^ 活率增加 ΗΑ 控制 ΜΤΧ/ΗΑ :6個月 對 ΗΑ 控制 5-FU/HA :6週及6個月 於 FU/HAHA 小鼠 研究顯示藉由降低 TDT，ΗΑ 發揮了 治療上的效力； ΗΑ降低淋巴結轉 移以及腫瘤形成。 (請先聞讀背面之注意事項再填寫本頁} 訂· 經濟部智慧財產局員工消費合作社印製 -72- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） 1284042 A7 B7 五、發明説明（7〇 ) (請先閲讀背面之注意事項再填寫本頁) 於該兩種研究中主要的差異在於該腫瘤的初始質量， 於該MTX/HA研究中該平均腫瘤體積為175.13mm3相 較於61.63 mm3於5-FU/HA研究。經由該粒子移動至 腫瘤中的動力以及一患者對腫瘤體積的反應，這可以說明 於相對於TDT估計之差異。 實例12典迭明蜇結合之治瘙法的瘙效 最常被用於人類初級與轉移的乳癌的治療療法是結合 化學治療法與環磷醯胺（Cyc)、MTX以及55-FU，其於 一 2 8日循環的第1日和第2 8日被投藥。該組合物療法通常 被稱作C M F，經常以6個循環被提供，於此時間該患者 的病況被再評估。該利用CMF治療法之抗腫瘤反應速度 已經被報導大約為50% (Bonadonna， 1981; Bonadonna，1 98 8)，但是該治療法有許多隨附的副作 用，例如疲倦、噁心、白血球減少以及嘔吐 (•Bonadonna，1976; Meyerowitz，1979)。由於利用 HA作為藥物用於ΜΤχ及5-FU輸送載劑之成功，我們評 經濟部智慧財產局員工消費合作社印製 估HA/CMF佐劑治療法超過6_週以及6個月治療療法的 療效與毒性。 MTX與5-FU分別如實例2及6中所述的被製備。該 cyc的儲備濃度藉由溶解lg的冷来乾燥的藥物於imi的 注射等級的無熱源蒸鶴水中並且以注射等級之〇 9%氣化 鈉補足成為5Gml。該儲備溶液被等分為小體積並且在使 用前與-20°C下冷凍。 -73- 1284042 A7 B7 五、發明説明（71 ) C M F注射藉由取出適量體積藥物儲備溶液被製備以 達成最終藥物濃度： 30mg/kg 5-FU (儲備溶液：20mg/ml) 15mg/kg MTX (儲備溶液：24.5mg/ml) 26mg/kg CYc (儲備溶液：20mg/ml) 12.5mg/kg HA (儲備溶液：i〇mg/ml) 為證實該C M F / Η A佐劑療法之療效以及任何可能的 毒性，於裸鼠中的人類乳癌腫瘤移植物被使用。為了儘可 能模擬治療療法，小鼠於一長期效用研究的6循環（6個月 )以及一短期效用研究的6循環（6週）中被治療。小鼠被隨 機分為7組每組8隻用於短期研究，以及$組每組8隻用於 長期研究。 小鼠以 30mg/kg 5-FU ; l5mg/kg ΜΤΧ ; 26mg/kg Cyc ±i2.5mg/kg於一 7日療法第1日與第6曰 被治療歷時6週，或是於一28日療法的第丨日與第8日被 治療歷時6個月。對照組包含有生理食鹽水、l2 5mg/kg HA的投藥，或是小鼠於第一曰有12 5mg/kg HA然後 於第2日有CMF。小鼠每日被檢視任何治療毒性，並且每 曰或每週量測腫瘤質量（參見表1丨）。 -74- 本紙張尺度適财類家標準（€刚八4規格（210'/297公釐） (請先閲讀背面之注意事項再填寫本頁} 衣 I訂 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 五、發明説明（ 經濟部智慧財產局員工消費合作社印製1284042 A7 _______B7_ V. Description of invention (69) Table! ! _: Use MTj^A and 5-FU/HA to the common results of the 瘙 瘙 method and the difference study similar results different results ΜΤΧ / ΗΑ : 6 months to 5-FU / HA · · 6 weeks to receive HA / Drug-treated mice showed significant weight gain; there was no significant increase in patient survival; inhibition of new tumor formation; presentation of a longer TDT; reduction of lymph node metastasis/ΗΑ: 6 months vs. 5-FU/HA: 6 Months showed a longer TDT 5-FU mice showed an increase in viability ΗΑ Control ΜΤΧ / ΗΑ : 6 months ΗΑ Control 5-FU / HA: 6 weeks and 6 months in FU / HAHA mice showed borrowing By reducing TDT, ΗΑ exerts therapeutic efficacy; ΗΑ reduces lymph node metastasis and tumor formation. (Please read the precautions on the back and fill out this page.) Order · Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -72- This paper scale applies to China National Standard (CNS) Α4 specification (210Χ297 mm) 1284042 A7 B7 , invention description (7〇) (please read the note on the back and then fill out this page) The main difference between the two studies is the initial mass of the tumor, which averaged 175.13mm3 in the MTX/HA study. Compared to 61.63 mm3 in the 5-FU/HA study, this can be explained by the difference in TTD estimates from the kinetics of the particles moving into the tumor and the response of a patient to the tumor volume. The treatment of breast cancer, which is most commonly used in human primary and metastatic breast cancer, is combined with chemotherapeutic treatment with cyclophosphamide (Cyc), MTX and 55-FU, which is the first in the 28th cycle. The composition is usually called CMF and is usually provided in 6 cycles, at which time the patient's condition is reassessed. The anti-tumor response rate using CMF therapy has been Report about It is 50% (Bonadonna, 1981; Bonadonna, 1 98 8), but the treatment has many accompanying side effects such as fatigue, nausea, white blood cell loss, and vomiting (•Bonadonna, 1976; Meyerowitz, 1979). The success of the drug in the use of sputum and 5-FU transport carrier, we evaluated the efficacy and toxicity of the treatment of HA/CMF adjuvant therapy over 6_week and 6 months of treatment by the Ministry of Economic Affairs' Intellectual Property Office employee consumption cooperative. Prepared as described in Examples 2 and 6 with 5-FU, respectively. The stock concentration of the cyc was prepared by dissolving lg of cold-dried drug in an imi-injected grade of non-pyrogenic steamed crane water and at an injection level of 〇9 % of sodium carbonate is made up to 5Gml. The stock solution is divided into small volumes and frozen at -20 ° C before use. -73- 1284042 A7 B7 V. Description of invention (71) CMF injection by taking a proper volume of drug The stock solution was prepared to achieve the final drug concentration: 30 mg/kg 5-FU (stock solution: 20 mg/ml) 15 mg/kg MTX (stock solution: 24.5 mg/ml) 26 mg/kg CYc (stock solution: 20 mg/ml) 12.5 Mg/kg HA (stock solution: i〇mg/ml) is To demonstrate the efficacy of this CMF / Η A adjuvant therapy and any possible toxicity, human breast cancer tumor grafts were used in nude mice. To mimic the treatment as much as possible, the mice were in 6 cycles of a long-term utility study (6 Month) and 6 cycles (6 weeks) of a short-term utility study were treated. Mice were randomized into 7 groups of 8 for each short-term study, and $8 for each group for long-term studies. The mice were treated with 30 mg/kg 5-FU; l5 mg/kg ΜΤΧ; 26 mg/kg Cyc ± i 2.5 mg/kg for the first day and the sixth sputum on the 7th day of treatment, 6 weeks, or a 28-day therapy. The third day and the eighth day were treated for 6 months. The control group contained physiological saline, 12 mg/kg HA, or the mice had 12 5 mg/kg HA in the first sputum and CMF on the 2nd day. Mice were examined daily for any therapeutic toxicity and tumor mass was measured weekly or weekly (see Table 1 丨). -74- This paper scales the standard for the financial class (€8-8 specifications (210'/297 mm) (please read the notes on the back and fill out this page). I am the Ministry of Economic Affairs, the Intellectual Property Bureau, the employee consumption cooperative. 1284042 A7 B7 V. Description of the Invention (Printed by the Consumers' Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs

表11 :治瘙投輳計割 治療群 服用量 6週研究： 6個月研究： 治療療法 1 ·生理食鹽 水 0 · 1 m 1 0 · 9 % 之 生理食鹽水（注 射等級） 7曰循環的1 及2 2 8曰循環的1 及8 2.CMF/HA 0.1 ml含有 ： 3 Omg/kg 5-FU+1 5mg/kg MTX + 26mg/kg Cy c+ 1 2.5 mg/kg HA 7曰循環的1 及2 2 8曰循環的1 及8 3. HA 0 · 1 m 1含有 ： 12.5mg/kg HA 7日循環的1 及2 2 8曰循環的1 及8 4.CMF 0 · 1 ml含有 ： 30mg/kg 5-FU 1 5mg/kg MTX + 26mg/kg C y c 7曰循環的1 及2 2 8曰循環的1 及8 5.HA然後 CMF 0.1 ml含有 ： 12.5 mg/kg 或 3 Omg/kg 5-FU 1 5mg/kg MTX + 26mg/kg Cyc 7曰循環 1 ： HA 2 ： 5-FU 3 ： HA 4 : 5-FU 2 8曰循環 1 ： HA 2 ： 5-FU 8 ： HA 9 : 5-FU 6. HA 0 · 1 m 1含有 ： 12.5mg/kg HA 7曰循環 1 ： HA 3 ·· HA -75- (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 1284042 Α7 Β7 五、發明説明（73 ) 如表12a以及12b中所示者，於該6週期間研究以下 結果被觀察到： 1).接受一含有HA (CMF/HA，HA接下來使用 C M F或僅使用η A)療法小鼠顯示增加的存活率，超過僅 使用CMF的群組50%。CMF治療群的平均存活時間為 40·4 days相對於所有其他群組的42中。 2 ) ·以Η A與C M F結合物治療的動物們顯示一顯著 的重置增加（t-test，Ρ<0·001)超過僅使用CMF或是ΗΑ 治療顯示一更好的結果（參見第2 2圖）。 3 ) ·以生理食鹽水治療之小鼠的腫瘤倍增時間（未治 療控制組）明顯的少於其他治療群（t_test，ρ<〇·〇()1)，但 是於其他治療群之間沒有統計學地顯著差異（參見第2 3 圖）。 4 ) · 於C M F以及C M F / Η Α治療群之間關於初級腫 瘤之終點體積有一顯著的差異，於此CM F於7/8的動物中 造成腫瘤質量的降低，而CMF/HA僅於3/8的動物中造成 腫瘤腫瘤體積的降低。 5 ) · 當小鼠以Η A 土 C M F來治療時，雖然僅以c M F 治療的小鼠顯示疲倦、結合膜炎、健康極差的徵候，並沒 有治療毒性被注意到。 當CMF/HA或ΗΑ治療法於28曰循環的第1曰及第8 曰被施用時，隨後的結果被發現；於治療群之間的存活時 間有一顯著差異 1). 於治療群之間於體重增加上沒有顯著差異。 -76- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210Χ297公釐） —Μ—^- —ϋϋ i··— ^ϋϋ I I (請先閱讀背面之注意事項再填寫本頁) I訂 經濟部智慧財產局員工消費合作社印製 1284042 五、發明説明（74 2) ·接文Η A士CMF之小鼠的初級的腫瘤顯示 的壞死與痂之形成 卞3)·於該初級腫瘤的治療中，CMF作為單一藥劑 /又有顯示超過生理舍趟匕 八Table 11: Treatment of sputum and sputum treatment group taking amount 6 weeks Study: 6 months study: Therapeutic therapy 1 · Physiological saline 0 · 1 m 1 0 · 9 % of physiological saline (injection grade) 7 曰 cycle 1 and 2 2 8 cycles of 1 and 8 2. CMF/HA 0.1 ml contains: 3 Omg/kg 5-FU+1 5mg/kg MTX + 26mg/kg Cy c+ 1 2.5 mg/kg HA 7曰 Cycle 1 And 2 2 8 曰 cycles of 1 and 8 3. HA 0 · 1 m 1 contains: 12.5 mg/kg HA 1 day and 2 2 8 曰 cycle of 1 and 8 4. CMF 0 · 1 ml contains: 30 mg /kg 5-FU 1 5mg/kg MTX + 26mg/kg C yc 7曰 Cycle 1 and 2 2 8曰 Cycles 1 and 8 5.HA then CMF 0.1 ml contains: 12.5 mg/kg or 3 Omg/kg 5 -FU 1 5 mg/kg MTX + 26 mg/kg Cyc 7 曰 Cycle 1: HA 2 : 5-FU 3 : HA 4 : 5-FU 2 8 曰 Cycle 1: HA 2 : 5-FU 8 : HA 9 : 5- FU 6. HA 0 · 1 m 1 contains: 12.5mg/kg HA 7曰 Cycle 1: HA 3 ·· HA -75- (Please read the notes on the back and fill out this page) This paper scale applies to Chinese national standards ( CNS ) A4 size (210X297 mm) 1284042 Α7 Β7 V. Invention description (73) as shown in Tables 12a and 12b The following results were observed during the 6 weeks: 1). Acceptance of a mouse containing HA (CMF/HA, HA followed by CMF or only η A) showed increased survival, exceeding the use of CMF alone. Group 50%. The mean survival time of the CMF treatment group was 40·4 days relative to 42 of all other groups. 2) • Animals treated with ΗA and CMF conjugates showed a significant increase in reset (t-test, Ρ<0·001) over a CMF only or ΗΑ treatment showed a better result (see section 2) 2 picture). 3) · The tumor doubling time (untreated control group) of mice treated with physiological saline was significantly less than that of other treatment groups (t_test, ρ<〇·〇()1), but there was no statistics between other treatment groups. Significant differences in learning (see Figure 2 3). 4) · There is a significant difference in the endpoint volume of primary tumors between CMF and CMF / Η Α treatment groups, where CM F causes a reduction in tumor mass in 7/8 animals, whereas CMF/HA is only 3/ A reduction in tumor tumor volume was caused in 8 animals. 5) · When mice were treated with ΗA soil C M F , although mice treated with c M F showed signs of fatigue, conjunctivitis, and poor health, no treatment toxicity was noted. When CMF/HA or sputum therapy was administered on Days 1 and 8 of the 28-inch cycle, subsequent results were found; there was a significant difference in survival between treatment groups 1). Between treatment groups There was no significant difference in weight gain. -76- This paper size applies to Chinese National Standard (CNS) Α4 specification (210Χ297 mm) —Μ—^- —ϋϋ i··— ^ϋϋ II (Please read the notes on the back and fill out this page) I Ministry of Intellectual Property Bureau employee consumption cooperative printed 1284042 V. Invention description (74 2) · 接文Η The primary tumor of A CMF mice shows necrosis and sputum formation 卞 3) · In the treatment of this primary tumor , CMF as a single agent / also shows more than physiological

艮皿水U及Η A治療的療效。添加Η A 至CMF產生—統計㈣貝著地較大的（卜卜以，㈣謝） 初級的腫瘤。 · 由此研究可以導出隨續的結論： 1) · HA/CMF佐劑治療法的長期的投藥（6個月）不 會顯示一療效的增加或是於治療副作用的降低。 2) · HA/CMF佐劑治療法的短期投藥（6_週）顯示 超過單獨以C M F治療的許多優點。 3 ) · 明顯的體重增加。 4 ) · 完成治療的動物增加5 0 %。 5).沒有諸如疲倦、結合膜炎、胃口不好等副作用 6) ·接受Η Α的小鼠沒有顯示任何毒性的記 號 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -77- 1284042The efficacy of the treatment of water U and Η A. Add Η A to CMF to produce - statistics (4) larger than the shell (Bu, (4) Xie) primary tumor. · This study can lead to subsequent conclusions: 1) • Long-term administration of HA/CMF adjuvant therapy (6 months) does not show an increase in efficacy or a reduction in side effects. 2) · Short-term administration of HA/CMF adjuvant therapy (6 weeks) shows many advantages over treatment with C M F alone. 3) · Significant weight gain. 4) · Increased animal mortality by 50%. 5). There are no side effects such as fatigue, membrane inflammation, and bad appetite. 6) · Mice receiving Η 没有 do not show any signs of toxicity (please read the notes on the back and fill out this page) Consumer Cooperative Printed -77-1284042

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、1T 本紙張尺度適用中國國家標準（CNS )八4規格（2丨0'乂297公釐） 1284042 A7 _________B7_ 五、發明説明（77 ) 實例13化學癃法的薷物典HA之本質的太質之NMRttf杳 為了進一步調查HA與化學療法的藥物之間的交互作 用，使用4核磁共振（NMR)光譜學。氧化氘（2h2〇 (99.96%))自 Cambridge Isotope Laboratories 取得 ° MTX 自 Faulding Pharmaceuticals 取得並且 5-FU 以及 HA儲備溶液如先前所述的被製備。 光譜在298 K下於一 Brtiker DRX光譜儀於6〇〇 MHz以一屏蔽梯度單元***作記錄。該2D實驗使用時間 比例相增量被記錄於一相-敏感模式的用以於ti維度正交 檢測（Marion & WUthrich，1 983)。該 2D 實驗使用 MLEV-17 spin lock sequence (Bax & Davis，1 985) 包含有TOCSY序列與120ms的混合時間；NOESY (Kumar et al·，1980) 250 以及 400 ms 的混合時間與 ROES Y光譜以一 250 ms的混合時間。探針的溫度校正藉 由與乙一醇化學的移位被達成。所有化學的移位（p p m)係 參考 4,4-二甲基-4-silapentane-l·磺酸鹽（DSS，0 ppm) 之曱基共振。 用於NOESY、ROESY以及T0CSY實驗之水訊息的 水溶液藉由使用改良的WATERGATE程序被達成 (Piotto et al·，1992)於此兩種1 ms的梯度脈波被應用 於一二項式3-9-19脈波的兩侧。於光譜6024 Hz與4096 複合的資料點被F2與512於F2維度的增量，以每增量 32掃描用於該T0CSY實驗以及80掃描用於N0ESY。 本紙張尺度適用中國國家標準（CNS ) A4規格（210X29_7^ ) ' J^i > .-I —ϋ —- - ϋ (請先閲讀背面之注意事項再填寫本頁) I訂 經濟部智慧財產局員工消費合作社印製 五、發明説明（78 ) NH 、子緩慢交換藉由獲取一系列的一維度（id)光譜獲 得超過16K資料點與使用32掃描。 NMR 擴散實驗於一 Briiker ΑΜχ 中 裝置以一於500 MHz操作的梯度控單元。所有的實驗以 16或64掃描於298 K超過16K的資料點與7575 Hz被 獲得。一 15.44 G/cm的梯度強度被用於該擴散實驗中。 母一擴散由一系列的12 PFGLED光譜中被獲得，於其 中該延遲（τ=20 ms，A=50 ms，T = 30 ms 以及 Te = 14 ms)並且於此該G之幅度被保持常數，但是該場梯度脈波 (8)被增量1 ms步驗由〇·2 1118至12.2 ms於該最終光譜 中〇 所有的光譜於一 Silicon Graphics Indigo, 1T This paper scale applies to China National Standard (CNS) VIII 4 specifications (2丨0'乂297 mm) 1284042 A7 _________B7_ V. Description of invention (77) Example 13 Chemical 癃 method 薷 薷 HA HA HA NMRttf杳 To further investigate the interaction between HA and chemotherapeutic drugs, 4-core magnetic resonance (NMR) spectroscopy was used. Cerium oxide (2h2〇 (99.96%)) was obtained from Cambridge Isotope Laboratories ° MTX was obtained from Faulding Pharmaceuticals and 5-FU and HA stock solutions were prepared as previously described. The spectra were recorded at 298 K on a Brtiker DRX spectrometer at 6 〇〇 MHz with a shielded gradient unit. The 2D experiment used time proportional phase increments were recorded in a phase-sensitive mode for orthogonal detection of the ti dimension (Marion & WUthrich, 1 983). The 2D experiment uses the MLEV-17 spin lock sequence (Bax & Davis, 1 985) to contain the TOCSY sequence with a mixing time of 120ms; NOESY (Kumar et al., 1980) 250 and 400 ms of mixing time and ROES Y spectrum. A mixing time of 250 ms. The temperature correction of the probe is achieved by chemical shifting with ethyl alcohol. All chemical shifts (p p m) are based on the sulfhydryl resonance of 4,4-dimethyl-4-silapentane-l·sulfonate (DSS, 0 ppm). The aqueous solutions for the water messages of the NOESY, ROESY and T0CSY experiments were achieved using the modified WATERGATE program (Piotto et al., 1992). These two 1 ms gradient pulses were applied to a binomial 3-9 -19 sides of the pulse wave. The data points composited at 6024 Hz and 4096 are incremented by F2 and 512 in the F2 dimension, with 32 scans per increment for the TOCSY experiment and 80 scans for NOESY. This paper scale applies to China National Standard (CNS) A4 specification (210X29_7^) ' J^i > .-I —ϋ —- - ϋ (Please read the note on the back and fill out this page) I Bureau employee consumption cooperative printing 5, invention description (78) NH, sub-slow exchange obtains more than 16K data points and uses 32 scans by obtaining a series of one-dimensional (id) spectra. The NMR diffusion experiment was performed in a Briiker® unit with a gradient control unit operating at 500 MHz. All experiments were performed with 16 or 64 scans at 298 K over 16 K data points and 7575 Hz. A gradient strength of 15.44 G/cm was used in this diffusion experiment. The mother-diffusion is obtained from a series of 12 PFGLED spectra where the delay (τ = 20 ms, A = 50 ms, T = 30 ms and Te = 14 ms) and where the amplitude of G is kept constant, However, the field gradient pulse (8) is incremented by 1 ms from 〇·2 1118 to 12.2 ms in the final spectrum. All spectra are in a Silicon Graphics Indigo

workstation 使用 Xwinr (Bruker)軟體處理。因為2D 實驗該t!維度係為以曰填滿至2〇48個真實數據點，並且 90°相移正弦鐘形鐘功效於F〇urier轉形前被應用。 經濟部智慧財產局員工消費合作社印製 该N M R貫驗被計用以顯示加入η A之Μ T X 1Η的 NMR光譜。藉由檢視該藥物ΜΤΧ之光譜的特定變化， 諸如峰之變寬或移動其可看出該藥物分子與ΗΑ是否有任 何交互作用。第、$24圖顯示ΜΤΧ溶於水中之1H NMR光譜 圖。此一光譜圖很快地辨認出於該氨甲喋呤中的每一個氫 原子基團。 氣甲唭呤 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X29^J^一 1284042 Α7 Β7 五、發明説明（79) i·* •一 M Jr— ί _一 · is n (請先閲讀背面之注意事項再填寫本頁) 氨甲嗓呤具有數個功能基團，其可潛在地與透明質 分子父互作用。該位於該2,4-胺基喋啶芳香環的MTX初 級胺類殘基可與該位於透明質分子上的羧基基團形成一個 離子結合。介於位於氨甲喋呤該胺類基團透明質之羧酸環 的經基基團之氫鍵交互作用係為另一可能。Μτχ之疏水 性芳香族環與折疊的透明質聚合物之疏水性碎片之間的疏 水***互作用亦為可能者。此等交互作用被圖式於第2 5 圖中。The workstation is processed using Xwinr (Bruker) software. Because of the 2D experiment, the t! dimension is filled with 2〇48 real data points with 曰, and the 90° phase-shifted sinusoidal bell clock is applied before the F〇urier transformation. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives. The N M R test was used to show the NMR spectrum of X T X 1Η added to η A . By examining the specific change in the spectrum of the drug, such as broadening or shifting the peak, it can be seen whether the drug molecule interacts with the sputum. The first and $24 graphs show the 1H NMR spectrum of hydrazine dissolved in water. This spectrogram quickly recognizes each hydrogen atom group in the methotrexate. The standard of the paper is applicable to the Chinese National Standard (CNS) Α4 specification (210X29^J^一1284042 Α7 Β7 5, invention description (79) i·* • a M Jr- ί _一· is n (please read first Precautions on the back side of this page) Methotrexate has several functional groups that can potentially interact with the parent of the hyaluronic molecule. The MTX primary amine in the 2,4-amino acridine aromatic ring The residue may form an ionic bond with the carboxyl group on the hyaluronan molecule. The hydrogen bond interaction between the radical group of the carboxylic acid ring located in the hyaluronan of the amine group is another possibility. Hydrophobic interactions between the hydrophobic aromatic ring of Μτχ and the hydrophobic fragments of the folded hyaluronic polymer are also possible. These interactions are illustrated in Figure 25.

、1T 為了處理氨甲喋呤與HA之間是否有一特定交互作用 的問題，該NMR實驗被設計用以顯示於加入ha之MTX 的1H NMR光譜的變化。第26圖顯示1該透明質酸在6〇〇 MHz以及298 Κ下之600 MHz光譜圖。 經濟部智慧財產局員工消費合作社印製 第27圖顯示MTX單獨以及MTX與增加的加入HA (50 kD a) 2毫微莫耳、1〇毫微莫耳、2〇毫微莫耳以 及80毫微莫耳於298 K下，該600 ΜΗζ β NMR光 譜圖。此等光譜圖顯示於氨甲喋呤該等峰沒有任何的化學 的移位位置變化。增加的峰呈現該Η Α的連續量的光譜圖 其元全與透明質酸的共振一致。由於於此光譜圖中Μτχ 的共振沒有化學的移位位置，其顯示該ΜΤχ分子沒有特 定的區域與ΗΑ分子產生交互作用。 決定在Μ ΤΧ之ΝΗ基團與該ΗΑ之酸基團之間是否有 一強交互作用的一種方法是量測該NJJ質子的交換速率。 該等氫原子傾向於並且可與該大量溶劑快速的交換。然而 右是，其等係與涉及一與ΗΑ分子之強烈反應而沒有被大1T To address the issue of whether there is a specific interaction between methotrexate and HA, the NMR experiment was designed to show the change in the 1H NMR spectrum of the MTX added to ha. Figure 26 shows a 600 MHz spectrum of the hyaluronic acid at 6 〇〇 MHz and 298 。. The Ministry of Economic Affairs Intellectual Property Office employee consumption cooperative printed on page 27 shows MTX alone and MTX with increased addition of HA (50 kD a) 2 nanomoles, 1 nanomole, 2 nanomole and 80 millimeters Micromolar at 298 K, the 600 ΜΗζ β NMR spectrum. These spectra show that the peaks do not have any chemical shift position changes in methotrexate. The increased peak exhibits a continuous amount of the spectrum of the enthalpy, which is consistent with the resonance of hyaluronic acid. Since the resonance of Μτχ in this spectrogram has no chemical shift position, it shows that there is no specific region of the ruthenium molecule that interacts with the ruthenium molecule. One method of determining whether there is a strong interaction between the hydrazine group and the acid group of the hydrazine is to measure the exchange rate of the NJJ proton. These hydrogen atoms tend to and can be rapidly exchanged with the large amount of solvent. However, the right is that it is not strongly affected by the strong reaction involving a molecule with a molecule.

1284042 A7 B7 五、發明説明（8〇)1284042 A7 B7 V. Description of invention (8〇)

It tT--__ (請先聞讀背面之注意事項再填寫本1) Ϊ溶液所保護’其等之交換速率會減少。於此等實驗中 MTX之胺類氫原子與來D2〇的說交換，其交換速率應該 提供一保護介於該MTX與HA之間。溶液NMR分析 藉由加入氧化氘至一 MTX及HA溶於0.5% w/v Na2C〇3 (pH 9)的溶液中被製備，其顯示在4分鐘内所有 MTX的胺類風原子已與樣品中的氣交換。此一結果暗示 位於Μ T X中的胺類氫原子與該大量溶劑無法避免交換。It tT--__ (please read the precautions on the back and fill in this 1). The exchange rate of the solution is reduced. In these experiments, the amine hydrogen atom of MTX is exchanged with D2, and the exchange rate should provide a protection between the MTX and HA. Solution NMR analysis was prepared by adding cerium oxide to a solution of MTX and HA dissolved in 0.5% w/v Na2C〇3 (pH 9), which showed that all of the amine wind atoms of MTX had been in the sample within 4 minutes. Gas exchange. This result suggests that the amine hydrogen atom located in Μ T X cannot be exchanged with the large amount of solvent.

、1T 該胺類氫峰自1 H NMR光譜圖消失的原因是因為氛 較氫具有一非常不同的共振，並且因此沒顯示於iH nmr 光譜圖中。相類似的實驗被例行的使用以測試於聚胜及蛋 白質中的骨架胺氩原子是否被大量溶劑所保護。當蛋白質 的醯胺氫原子涉及該氫鍵排列其安定一 α ·螺旋以及石-平 板二級結構該等氫原子的交換速率經常會戲劇性地減少。 於某些案例中該氫訊息涉及此等交互作用其可堅持數小時 數日甚至數個月端視該交互作用的強度以及其由大量溶液 的保護程度（例如於一疏水性蛋質的核心中）。 經濟部智慧財產局員工消費合作社印製 擴散實驗被ΜΤΧ單獨的，以及於ΗΑ出現下的執行。 此等實驗可以指出是否被夾帶的ΜΤΧ之擴散由於ΗΑ篩網 的出現而被延遲。該1H NMR擴散實驗的重覆指出對於 ΜΤΧ分子的主要部分並沒有μτχ擴散速度的延遲因為於 該ΜΤΧ與Η Α存在的ΜΤΧ之間的擴散係數有一可忽略的 差異。此一發現可以暗示於Η A網絡中在Η A分子之間有 大量的溶液腔其容許藥物於其間自由的擴散至基質中。 1284042 A7 B7 五、發明説明（81 ) ~~~ 該擴散實驗暗示大部分的Μτχ分子係自由的經由ha 分子被擴散。此-腳本並沒有被列入考量，若是一小部分 的（也就是5%ΜΤΧ分子）以一不特定的方式與ΗΑ分子交 互作用。 由前述實驗ΜΤΧ與ΗΑ沒有強烈地交互作用是很明 顯的，若有任何交互作用也是不特定的。用以測試弱連結 (10 3-10 7 Μ)的一種方法是測試用於不特定的結合是執 行一轉移NOESY實驗於此等2D實驗中越峰將顯示該連結 是否微弱地結合至HA。一轉移的MTX/HA iN〇ESY光 譜圖顯示沒有越峰因為ΜΤΧ結合至HA其暗示了在Μτχ 與HA之間一可忽略的交互作用。 如同一進一步的確定是否小部分的（亦即5 %的μTX 分子）以一不特定的方式微弱地與Η Α分子交互作用。於 ROES Y光譜圖中的峰應該顯示是否有一即使是小部分的 ΜTX分子於自由的及結合至HA的狀態間化學的交換。 A 250 ms R0ESY光谱圖沒有顯不任何地化學的交換峰 其暗示即使是小百分比的MTX也沒有與HA交互作用。 5-氟尿嘧啶 第 28 圖顯示該 5-FU 及 5-FU (1.25 mg/mL，1.6 mg/mL and 6.4 mg/mL) with HA (750 kDa, 3 mg/mL)在70.0與8.5 ppm之間，於298 K下的起始實 驗之600 MHz β NMR光譜，以調查是否有一交互作 用與50kDa的HA被執行。在5-FU與HA沒有交互作用 ^紙張尺度適用中國國家標準（CNS ) Α4規格（ m.-llr-J^i eti *，—ϋ —>^1 —1 1 (請先閱讀背面之注意事項再填寫本頁)The reason why the amine hydrogen peak disappeared from the 1 H NMR spectrum is because the atmosphere has a very different resonance than hydrogen and is therefore not shown in the iH nmr spectrum. A similar experiment was routinely used to test whether the backbone amine argon atoms in the polysulfon and protein were protected by a large amount of solvent. When the indoleamine hydrogen atom of a protein is involved in the hydrogen bond arrangement, it stabilizes an α·helix and the stone-plate secondary structure. The exchange rate of such hydrogen atoms is often dramatically reduced. In some cases, the hydrogen message relates to such interactions that can persist for hours, even months, depending on the strength of the interaction and its degree of protection from the bulk of the solution (eg, in the core of a hydrophobic egg) ). Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives. The diffusion experiment was carried out separately and executed under the presence of ΗΑ. These experiments can indicate whether the spread of the entrained helium is delayed due to the presence of the helium screen. The repetition of this 1H NMR diffusion experiment indicates that there is no delay in the diffusion rate of μτχ for the major portion of the ruthenium molecule because there is a negligible difference in the diffusion coefficient between the ruthenium and the ruthenium present. This finding may imply that there is a large amount of solution chamber between the ΗA molecules in the ΗA network which allows the drug to diffuse freely into the matrix therebetween. 1284042 A7 B7 V. INSTRUCTIONS (81) ~~~ This diffusion experiment suggests that most of the Μτχ molecules are free to diffuse through the ha molecule. This - script is not considered, if a small part (that is, 5% ΜΤΧ molecule) interacts with the ΗΑ molecule in an unspecified way. It is obvious that there is no strong interaction between the aforementioned experiments and ΗΑ, and any interaction is not specific. One method used to test weak linkages (10 3-10 7 Μ) is to test for unspecific binding to perform a transfer NOESY experiment. In such 2D experiments, Yuefeng will show whether the link is weakly bound to HA. A transferred MTX/HA iN〇ESY spectrogram shows no peak because the binding of ruthenium to HA suggests a negligible interaction between Μτχ and HA. As a further determination, it is determined whether a small portion (i.e., 5% of the μTX molecules) interacts weakly with the Η molecule in an unspecified manner. The peaks in the ROES Y spectrum should show whether there is a chemical exchange between even a small fraction of the ΜTX molecules in a state of free and binding to HA. The A 250 ms R0ESY spectrum shows no apparent chemical exchange peaks, suggesting that even a small percentage of MTX does not interact with HA. 5-fluorouracil Figure 28 shows that the 5-FU and 5-FU (1.25 mg/mL, 1.6 mg/mL and 6.4 mg/mL) with HA (750 kDa, 3 mg/mL) are between 70.0 and 8.5 ppm, A 600 MHz beta NMR spectrum of the initial experiment at 298 K was investigated to investigate whether an interaction was performed with a 50 kDa HA. There is no interaction between 5-FU and HA. Paper scale applies to Chinese National Standard (CNS) Α4 specification ( m.-llr-J^i eti *, —ϋ —>^1 —1 1 (Please read the note on the back first) Please fill out this page again)

、1T 經濟部智慧財產局員工消費合作社印製 1284042 A7 B7 重 擴 五、發明説明（82 被觀察到（資料未顯示）。為了調查一交互作用是否視Η A 的分子量而定，進一步的滴定實驗與750 kDa透明質被 執行。該被使用的5-FU濃度相等於HA/5-FU佐劑治療 法之Kings College London配方研究其入一製劑用於 瑞士的臨床的階段。該濃度被設計以模擬該用於混合 HA/5-FU的濃度，於該注入袋中以及ha/5-FU濃度於 血漿中被估計。不幸地，該5 ·氟尿嘧啶的胺類共振與此 等配方之大量的溶劑水在pH (8.8 to 9.1)快速交換因此 此等共振於5-FU的光譜圖中係為不可見者。僅有一個CH 的共振於5-FU的光譜圖中係為可見者。降低此等溶液之 pH係為不可行的，因為5_FU於低1)^1將會沉澱。此等光 谱圖顯示於該化學5-FU之峰之的移位位置並沒有改變。 此等光譜指出該5-FU沒有顯示與HA分子交互作用。 擴散實驗於5-FU單獨以及於HA的存在下被執行c 覆1H NMR擴散實驗的結果顯示於表13中指出 散藉由透明質的出現沒有任何遲延，因為於5_叩單獨^ 5-FU於HA存在之擴散係數有一可忽略的差異。 、 1_ιϋ^·Μ·Ηί ϋ— ϋϋ eKKmmmmmm —ϋ (請先閲讀背面之注意事項再填寫本頁) 訂 經濟部智慧財產局員工消費合作社印製1T Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1240442 A7 B7 re-expansion five, invention description (82 observed (data not shown). In order to investigate whether an interaction depends on the molecular weight of A, further titration experiment Performed with 750 kDa of hyaluronic acid. The 5-FU concentration used was equivalent to the HA/5-FU adjuvant treatment of the Kings College London formulation study for its clinical use in a formulation for use in Switzerland. The concentration used to mix the HA/5-FU was simulated and estimated in the infusion bag and the ha/5-FU concentration in plasma. Unfortunately, the amine resonance of the 5-fluorouracil and the large amount of solvent of these formulations The rapid exchange of water at pH (8.8 to 9.1) is therefore invisible in the spectra of these resonances in 5-FU. Only one resonance of the CH is visible in the 5-FU spectrum. The pH of the solution is not feasible because 5_FU will precipitate at a low level of 1). These spectra show no shift in the shift position of the peak of the chemical 5-FU. These spectra indicate that the 5-FU does not show interaction with HA molecules. Diffusion experiments were performed on 5-FU alone and in the presence of HA. The results of the c-cladding 1H NMR diffusion experiments are shown in Table 13 indicating that there is no delay in the presence of hyalin, since 5_叩 alone ^ 5-FU There is a negligible difference in the diffusion coefficient present in HA. , 1_ιϋ^·Μ·Ηί ϋ—ϋϋ eKKmmmmmm —ϋ (Please read the notes on the back and fill out this page) Order Printed by the Ministry of Economic Affairs Intellectual Property Office Staff Cooperatives

1284042 A7 _________ 五、發明説明（83 ) U : NMR擴散值數 樣 品 HA儲備 (750kDa, 1 Omg/mL) 5-FU (2 Omg/mL ) 擴散常數m2/s 4 62.5 β L 9.245χ101ϋ/8.900χ10'10 1 300 β L 62.5 β L 8.701χ1 0*10/9.030χ1〇·10 5 i〇 /z L 9.141xl010/9.204xl010 2 300 β L SO βL 8·948χ10·1ο/9·021χ10_10 6 - 320 β L 9.004x1 0'10/8.997xl〇·10 3 300 β L 320 β L 8.851xl〇-10/8.833xl〇·10 2D實驗、NOESY以及ROESY : 2D實驗於5_FU無 法進行，因為只有一種共振於其光譜中係可見者。 於透明質存在下的MTX以及5-FU之NMR分析使用 滴定實驗、氘交換實驗以及用於5-FU、擴散實驗已經顯 示在化學療法藥物與透明質之間沒有任何的交互作用。此 等結果暗示化學療法的藥物藉由透明質網絡之夾帶足以增 加藥物輸送至病理的位置的數量。此等結果完全與先前使 用凝膠過濾色層分析法、平衡透析一致，並且透明質與 MTX及5FU之間的分子交互作用之cd光譜也沒有檢測 到一交互作用。 ------ (請先閲讀背面之注意事項再填寫本頁) I訂 經濟部智慧財產局員工消費合作社印製 本紙張尺度適财酬家標準（CNS ) A4規格（2i〇xU^ ) 1284042 A7 B7 五、發明説明（84) 參老文獻 (請先閱讀背面之注意事項再填寫本頁)1284042 A7 _________ V. INSTRUCTIONS (83) U : NMR diffusion value sample HA reserve (750 kDa, 1 Omg/mL) 5-FU (2 Omg/mL) Diffusion constant m2/s 4 62.5 β L 9.245χ101ϋ/8.900χ10 '10 1 300 β L 62.5 β L 8.701χ1 0*10/9.030χ1〇·10 5 i〇/z L 9.141xl010/9.204xl010 2 300 β L SO βL 8·948χ10·1ο/9·021χ10_10 6 - 320 β L 9.004x1 0'10/8.997xl〇·10 3 300 β L 320 β L 8.851xl〇-10/8.833xl〇·10 2D experiment, NOESY and ROESY: 2D experiment cannot be performed in 5_FU because there is only one resonance in it Visible in the spectrum. NMR analysis of MTX and 5-FU in the presence of hyaluromes using titration experiments, hydrazine exchange experiments, and for 5-FU, diffusion experiments have shown no interaction between chemotherapeutic drugs and hyaluronan. These results suggest that the drug of the chemotherapeutic agent is entrained by the hyaluronan network to increase the number of sites where the drug is delivered to the pathology. These results were completely consistent with the previously used gel filtration chromatography, equilibrium dialysis, and no interaction was detected with the cd spectra of the molecular interaction between hyaluronan and MTX and 5FU. ------ (Please read the notes on the back and fill out this page.) I. Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, Printed Paper Scale, Appropriation for Money (CNS) A4 Specification (2i〇xU^) 1284042 A7 B7 V. INSTRUCTIONS (84) Participate in the old literature (please read the notes on the back and fill out this page)

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Bax, A. & Davis, D.G. ( 1 985) MLEV-17 based two-dimensional homo-nuclear magnetization transfer spectroscopy. J. Magn. Reson. 65，3 5 5-360.Bax, A. & Davis, D.G. (1 985) MLEV-17 based two-dimensional homo-nuclear magnetization transfer spectroscopy. J. Magn. Reson. 65,3 5 5-360.

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Bisset D，Setanioans A, Cassidy J，Graham MA and Kerr DJ， 1 9 9 3. Cancer Res 53:523-527. 本紙張尺度適用中國國家標準（CNS ) A4規格（21(^2^7¾¾ ) 1284042 A7 B7 五、發明説明（85Bisset D, Setanioans A, Cassidy J, Graham MA and Kerr DJ, 1 9 9 3. Cancer Res 53: 523-527. This paper scale applies to the Chinese National Standard (CNS) A4 specification (21(^2^73⁄43⁄4) 1284042 A7 B7 V. Description of invention (85

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Carter D, 1990. Interpretation of Breast Biopsies，Second Edition Raven Press, New York. 請 先 閲 % 背 之 注 項 再 填 寫 本 頁 經濟部智慧財產局員工消費合作社印製Carter D, 1990. Interpretation of Breast Biopsies, Second Edition Raven Press, New York. Please read the % back note and fill out this page. Printed by the Intellectual Property Office of the Ministry of Economic Affairs.

Coradini, D·，Pellizzaro，C·，Miglierini， G.,Daidone, M.G. and Perbellini, A. (1 999) Hyaluronic Acid As Drug Delivery For Sodium Butyrate: Improvement Of The Anti-Proliferative Activity On A Breast-Cancer Cell Line. Int. J· Cancer· 81 :pp.411-416 Culty M, Nguyen HA and Underhill CB, 19 92. J Cell Biol 1 1 6(4) : 1 05 5-1 062 . Culty, M., Shizari, M., Erik, W., Thompson, and Underhill, C.B. (1994) Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential. Journal of Cellular Physiology 160: pp 275-286 本紙張尺度適用中國國家標準（CNS ) A4規格（210'乂2§|螯） 1284042 A7 B7 五、發明説明（86) (請先閲讀背面之注意事項再填寫本頁)Coradini, D., Pellizzaro, C., Miglierini, G., Daidone, MG and Perbellini, A. (1 999) Hyaluronic Acid As Drug Delivery For Sodium Butyrate: Improvement Of The Anti-Proliferative Activity On A Breast-Cancer Cell Line Int. J· Cancer· 81 : pp. 411-416 Culty M, Nguyen HA and Underhill CB, 19 92. J Cell Biol 1 1 6(4) : 1 05 5-1 062 . Culty, M., Shizari, M., Erik, W., Thompson, and Underhill, CB (1994) Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: Correlation with invasive potential. Journal of Cellular Physiology 160: pp 275-286 Paper scale applies to Chinese National Standard (CNS) A4 specification (210'乂2§|chelating) 1284042 A7 B7 V. Invention description (86) (Please read the note on the back and fill out this page)

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Eriksson, S·，Fraser，JRE·，Laurent，TC·，Perioft， H. and Smedsrod，B. ( 1 983) Endothelia cells are a site of uptake and degradation of hyaluronic acid in the liver. Exp Cell Res · 144: pp. 223-228Eriksson, S., Fraser, JRE, Laurent, TC, Perioft, H. and Smedsrod, B. (1 983) Endothelia cells are a site of uptake and degradation of hyaluronic acid in the liver. Exp Cell Res · 144: Pp. 223-228

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Friedrichs G, Folker HJ, A-rlt PA and Gunthert U, 1995. The Lancet 345:1237-1238. 經濟部智慧財產局員工消費合作社印製Friedrichs G, Folker HJ, A-rlt PA and Gunthert U, 1995. The Lancet 345:1237-1238. Printed by the Intellectual Property Office of the Ministry of Economic Affairs

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Gustafson S，Bjorkman T, Forsberg N，Lind T， Wikstrom T and Lidholt K, (1 995a). Accessible hyaluronan receptors identical to ICAM-1 in mouse mast tumour cells. Glycoconjugate J. 12，3 5 0-3 5 5. 本紙張尺度適用中國國家標準（CNS ) A4規格（210X2^1^ ) 1284042 A7 B7 五、發明説明（87)Gustafson S, Bjorkman T, Forsberg N, Lind T, Wikstrom T and Lidholt K, (1 995a). Accessible hyaluronan receptors identical to ICAM-1 in mouse mast tumour cells. Glycoconjugate J. 12,3 5 0-3 5 5. This paper scale applies to China National Standard (CNS) A4 specification (210X2^1^) 1284042 A7 B7 V. Description of invention (87)

Gustafon，S·，Bj6rkman，T_，Forsberg， (請先閲讀背面之注意事項再填寫本頁) N.，McCourt，P·，Wikstr6m，T·，Lilja，K·，Tinner，B·， Fuxe，K·，Westlin，J.-E·，Lidholt，K. Lind，T·， Westerberg，G·，Bergstrom，M，Langstrom，B·，de la Torre，M·，Bergh，J·，Hagberg，H·，Glimelius，B·， Lindahl, U., & Laurent, T.C. (1 995b) studies on receptors for hyaluronan and the turnover of radio actively-lab e lied hyaluronan in mice and rats. Royal Soc. Med. Round Table Series no36, 5-7.Gustafon, S·, Bj6rkman, T_, Forsberg, (please read the notes on the back and fill out this page) N., McCourt, P·, Wikstr6m, T·, Lilja, K·, Tinner, B·, Fuxe, K ·, Westlin, J.-E., Lidholt, K. Lind, T., Westerberg, G., Bergstrom, M, Langstrom, B., de la Torre, M., Bergh, J., Hagberg, H., Glimelius, B., Lindahl, U., & Laurent, TC (1 995b) studies on receptors for hyaluronan and the turnover of radio actively-lab e lied hyaluronan in mice and rats. Royal Soc. Med. Round Table Series no36, 5-7.

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Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. Cell 82: pp 19-28Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. Cell 82: pp 19-28

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本紙張尺度適用中國國家標準（CNS ) A4規格（210/:^7¾¾ )This paper scale applies to the Chinese National Standard (CNS) A4 specification (210/:^73⁄43⁄4)

Claims (1)

12840 斤月7 Et修(/)正本 六、申請專利範圍 第089100433號專利再審查案申請專利範圍修正本 修正日期：95年8月 1· 一種針對透明質（hyaluronan)與一細胞毒性或抗贅生藥 劑的組合用於製造一藥物之用途，該藥物係供用於治療 一選自於癌症、腫瘤與贅瘤之疾病，其中該藥劑的癌細 胞殺滅潛力會由於該透明質與該藥劑的組合使用而被 增強。 2·如申請專利範圍第丨項之用途，其中該藥劑係擇自於下 列所構成之群組：氨甲嘌呤、佩里塔克爾[paclhaxel(紅 豆杉醇）]、5-氟尿嘧啶、環磷醯胺，以及此等之組合。 3 ·種細胞毒性或抗贅生藥學組成物，其包含透明質 (hyaluronan)以及一細胞毒性或抗贅生藥劑，其中該組成 物係供用於全身性投藥。 4·如申請專利範圍第1項之針對透明質（hyalur〇nan)與一 細胞毒性或抗贅生藥劑的組合用於製造一藥物之用 途，該藥物係供用於治療一選自於癌症、腫瘤與贅瘤 之疾病’其中該癌症、腫瘤與贅瘤之疾病係具藥物抗 性。 5·如申請專利範圍第1項之針對透明質（hyalur〇nan)與一 細胞毒性或抗贅生藥劑的組合用於製造一藥物之用 途，忒藥物係供用於治療一選自於癌症、腫瘤與贅瘤 之疾病，其中該透明質（hyalur〇nan)係可夾帶及/或結合 一抗贅生或細胞毒性藥劑。 96 1284042 六、申清專利範圍 6·如申睛專利範圍第5項之用途，其中該癌症、腫瘤與贅 瘤之疾病係具藥物抗性。 7· 士申明專利範圍苐6項之針對透明質（hyaluronan)與一 細胞毒性或抗贅生藥劑的組合用於製造一藥物之用 途’該藥物係供用於治療一選自於癌症、腫瘤與贅瘤 之疾病其中邊透明質（hyaluronan)係可結合至在該癌 症、腫瘤與贅瘤細胞上的受體。 8 · 4申明專利範圍弟4項之針對透明質（hyalUr〇nan)與一 細胞毋性或抗贅生藥劑的組合用於製造一藥物之用 途，該藥物係供用於治療一具藥物抗性之癌症、腫瘤 與資瘤疾病’其中該透明質（hyaluronan)增強該藥劑之 有效性，藉由提供該藥劑於一個體體内之一更長的曝 露期間，相較於單獨使用該藥劑。 士申#專利範圍第4項之針對透明質（hyaluronan)與一 細胞毒性或抗贅生藥劑的組合用於製造一藥物之用 途，該藥物係供用於治療一具藥物抗性之癌症、腫瘤 與贅瘤疾病，其中該癌症、腫瘤與贅瘤在單獨使用該 細胞毒性或抗贅生藥劑時係對該組合中之細胞毒性或 抗贅生藥劑具有抗性。 10·如申請專利範圍第8項之用途，其中該癌症係對於擇自 於由氨曱嘌呤、佩里塔克爾[paclitaxel(紅豆杉醇）]、5_ 氟尿嘧啶、環磷醯胺，以及此等之組合物所構成之群 組的一種或多種藥劑具有抗性。 1 · 士申《月專利範圍弟4項之針對透明質（hyalUr〇nan)與— 97 1284042 六、申請專利範圍 細胞毒性或抗贅生藥劑的組合用於製造一藥物之用 途，該藥物係供用於治療一具藥物抗性之癌症、腫瘤 與贅瘤疾病，其中相較於單獨投與該藥劑，該藥物降 低被投與至一個體體内之該藥劑所引起的腸胃道毒 性。 9812840 kg month 7 Et repair (/) original six, the patent application scope 089100433 patent re-examination application patent scope amendment This amendment date: August 1st, 1995 · One for hyaluronan and a cytotoxic or anti-caries A combination of biopharmaceuticals for the manufacture of a medicament for the treatment of a disease selected from the group consisting of cancer, tumor and neoplasm, wherein the cancer cell killing potential of the agent is due to the combination of the hyaluronan and the agent It is enhanced by use. 2. The use of the scope of the patent application, wherein the agent is selected from the group consisting of methotrexate, peritaxel [paclhaxel], 5-fluorouracil, cyclophosphazene Amines, and combinations of these. 3. A cytotoxic or anti-neoplastic pharmaceutical composition comprising hyaluronan and a cytotoxic or anti-neoplastic agent, wherein the composition is for systemic administration. 4. The use of a combination of hyalurin and a cytotoxic or anti-neoplastic agent for the manufacture of a medicament for use in the treatment of a cancer selected from the group consisting of cancer and tumors. A disease associated with a tumor, in which the cancer, tumor, and tumor are drug resistant. 5. The use of a combination of hyaluran and a cytotoxic or anti-neoplastic agent for the manufacture of a medicament, as claimed in claim 1, is for use in the treatment of a cancer, tumor. A disease associated with a tumor, wherein the hyalurin can bind and/or bind an anti-neoplastic or cytotoxic agent. 96 1284042 6. The scope of patent application of Shenqing 6. The use of the fifth paragraph of the scope of the patent application, wherein the cancer, tumor and tumor disease are drug resistant. 7. The scope of the patent scope 苐6 for hyaluronan and a combination of a cytotoxic or anti-neoplastic agent for the manufacture of a drug for treatment of a cancer, tumor and sputum A disease of the tumor in which the hyaluronan line binds to receptors on the cancer, tumor and tumor cells. 8 · 4 claims the patent scope of the fourth of the combination of hyaluronic acid (hyalUr〇nan) and a cell-based or anti-neoplastic agent for the manufacture of a drug for the treatment of a drug resistance Cancer, tumor and tumor-bearing disease 'where the hyaluronan enhances the effectiveness of the agent by providing the agent for a longer exposure period in one body than when the agent is used alone. The combination of hyaluronan and a cytotoxic or anti-neoplastic agent for the manufacture of a drug for the treatment of a drug-resistant cancer, tumor and A neoplastic disease wherein the cancer, tumor, and neoplasm are resistant to cytotoxic or anti-neoplastic agents in the combination when the cytotoxic or anti-neoplastic agent is used alone. 10. The use of claim 8 wherein the cancer is selected from the group consisting of aminoguanidine, pacitaxel (caclitaxel), 5 fluorouracil, cyclophosphamide, and the like One or more agents of the group of compositions are resistant. 1 · Shishen's “Monthly Patent Range 4 for Hyalin (hyalUr〇nan) and – 97 1284042 VI. Application for a combination of cytotoxic or anti-neoplastic agents for the manufacture of a drug for use For the treatment of a drug-resistant cancer, tumor and neoplasm disease, wherein the drug reduces the gastrointestinal toxicity caused by the agent administered to a body as compared to the administration of the agent alone. 98